The physiological roles of mycorrhizal fungi, including arbuscular mycorrhizal fungi on seedlings and nut trees, are increasingly studied. Mycorrhizal fungi provide beneficial symbiosis in the roots of nut trees, accelerate plant growth and biomass production, nutrient acquisition, and increase potential tolerance to abiotic stress, e.g., drought and salt tolerance. Consequently, future outlooks in this field seem optimistic.In pistachio, several fungal and bacterial diseases can infest both the above-ground and under-ground tree parts. Among these, Phytophthora root and crown rot , Armillaria root rot , and Verticillium wilt are the three most serious soil borne fungal diseases of pistachio trees worldwide. The P. vera is the only pistachio species that produces edible nuts large enough for commercial use . Initial evaluations demonstrated that the P. vera seedling trees were susceptible to soilborne pathogens Phytophthora spp.; Verticillium dahlia, and nematodes.Therefore, other available Pistacia spp. were used as root stocks. Verticillium wilt has killed a majority of the trees in the late 1970s and caused growers the most severe economic losses ever experienced in California. A small number of P. integerrima seedlings from an Iranian seedling tree selected and planted at the USDA Plant Introduction Station in Chico were found to be tolerant to Verticillium wilt; the trees can be infested but exhibit few symptoms and no mortality. This P. integerrima seedling root stock was quickly commercialized as Pioneer Gold 1 . Verticillium-tolerant P. integerrima was then used to produce UCB1 which is moderately resistant to this disease; it exhibits mild symptoms when infested but, as with PGI, no mortality. The P. atlantica and P. terebinthus root stocks are susceptible to Verticillium wilt . Armillaria root rot occasionally affects pistachio and resistant root stocks would offer the best protection.

Field trials indicate that P. terebinthus and UCB1 are tolerant,blueberry plant pot whereas P. atlantica and P. integerrima are susceptible to this pathogen. Root and crown rot caused by Phytophthora spp. also can affect pistachio trees. According to Ferguson et al., UCB1 and P. atlantica are more tolerant to Phytophthora root and crown rot than P. integerima. Epstein et al. studied the resistance of four root stocks to Verticillium dahlia. Yield, growth, incidence of Verticillium symptoms, and mortality rates were studied for 10 consecutive years. UCB1 and P. integerrima showed the greatest tree vigor, and UCB1 had the fewest symptoms. UCB1 is also resistant to Phytophthora. Thus, UCB1 has become the major root stock in California. However, a stunted and difficult-to-graft phenotype has emerged in California in clonally reproduced UCB1 root stocks from multiple sources. This has been at times, since 2010, a serious production problem which has been variously attributed to either somaclonal mutation during in vitro propagation or to Rhodoccus sp. bacterial infection, Chang et al.. The syndrome was identified by its appearance as Pistachio Bushy Top Syndrome, PBTS. However, as this problem proved to be non-transmissible in the field, and nurseries can now identify it in young root stocks, it is no longer a problem. Nouri et al. reported a new pathogen, Macrophomina phaseolina in Kern County of California which is characterized by wilted foliage combined with crown rot of the root stock. UCB1 is highly susceptible to M. phaseolina and this pathogen is now an emerging threat to pistachio production in California. One important biotic stress in Persian walnut is black line disease caused by Cherry leaf roll virus. Persian walnut tolerates this virus and is generally symptomless. In contrast, J. hindsii or its hybrids are resistant to CLRV. Blackline symptoms occur when a hypersensitive root stock [Northern California Black walnut , other black walnuts, or hybrids of these with J. regia, are used as root stock for Persian walnut. The virus is transmitted through infected pollen and scions. The pollenborne virus enters through flowers during pollination and is systemically transported to the graft union. The resulting hypersensitive reaction of the root stock and death of tissue at the graft union blocks nutrient and water transport between the root stock and scion.

The hypersensitive response to this virus is controlled by a single dominant gene. To develop CLRV-resistant scion cultivars capable of blocking the virus at the pistillate flower and/or movement toward the graft union, a breeding program was initiated in 1984 the University of California-Davis to backcross resistance from Paradox into scion cultivars with commercially acceptable horticultural traits. This program is still ongoing. A DNA marker related to CLRV-resistance that maps to ~6.2 Mb onchromosome 14 has been developed in order to accelerate selection of CLRV-resistant offspring. In continuation of work started by E. Germain , a hybrid resistant to black line is in evaluation to be registered in France. In California, screening of a huge multi-species Juglans population, J. regia, J. microcarpa, J. major, J. cathayensis, and others and targeted interspecies hybridization between the selected superior genotypes to produce root stocks resistant to the soil borne pathogens, Agrobacterium tumefaciens, Phytophthora spp.; Pratylenchus vulnus, and Armillaria mellea, has been in progress for several years and is continuing. Crown gall is a major root stock issue in walnuts, particularly when using Paradox hybrid root stocks. This bacterial disease can significantly reduce production and increase management costs. The RNAi technology, RNA interference has been used experimentally to suppress genes involved in the plant response to the bacterium. Silencing of tryptophan monooxygenase and isopentenyl transferase genes blocks bacterial induction of de novo auxin and cytokinin and therefore prevents gall development. Using RNAi-mediated silencing technology, walnut researchers at UC-Davis were also able to develop apparent nematode resistance in Paradox microshoots evaluated in vitro but this work has not been confirmed in the greenhouse or field trials. Nematodes are another serious problem for nut growers. Three separate root-knot nematode resistance genes have been identified in Prunus species, Ma in the Myrobalan plum clones ‘P29800 and ‘P21750 , RMia in the peach root stock ‘Nemared’, and RMja in the bitter almond ‘Alnem’. Pyramiding of these three genes by interspecific crosses of almond × peach × Myrobalan is the main objective of the French root stock breeding program. 

To ensure the presence of the three genes in the same root stock, it has been necessary to develop effective molecular markers. The identification of intra-gene markers for nematode-resistance genes Ma and RMia has allowed the application of marker-assisted selection for these two genes. The RMja gene is located on linkage group 7 of the Prunus genome in the same region as the Ma gene. Chestnut cultivation has been threatened by chestnut blight and root rot diseases. The first pandemic disease for chestnut was root rot. Today two species of root rot are widelyspread in Europe and Asia Minor. The most effective method against root rot disease is using resistant root stocks. Due to the resistance to root rot, C. crenata and C. mollissima trees were imported into Europe at the beginning of the 19th century. The following years showed that their nut quality was low, and they were sensitive to spring frosts. Therefore, they were used as root stocks. However, graft incompatibility was observed. In France, two of these genotypes were registered as ‘Ipharra’ and ‘Marki’. Schad et al. planted some superior genotypes in orchards infected with Phytophthora spp. in 1946. As a result of this study, natural hybrids of C. crenata × C. sativa were obtained [260,261]. Amongst those, ‘Marsol’ and ‘Maraval’ have been used as resistant root stocks. ‘Marigoule’ has been used in forest areas due to its fast-growing characteristic. Now, ‘Marigoule’ is also used as root stocks in many countries due to its resistance to root rot and tolerance to the chestnut blight . However, seedlings of the ‘Marigoule’ are not tolerant or resistant to these diseases as a scion cultivar. Ten years of observation have demonstrated that ‘Marigoule’ seedling survival from root rot is only 10% greater than European chestnut seedlings. As a continuation of these studies, in 1980,plastic gardening pots a new breeding program was initiated in France. Early results showed that ‘Maridonne’ and ‘Marlhac’ root stocks could also be used against root rot . This breeding program is continuing. A similar program was also initiated in Spain by Gallasteguie in 1926 and continued by Urquijo. They imported some chestnut genotypes from Korea and Japan between 1917 and 1940. In this study, 263,000 genotypes were tested and 12,000 of these were found resistant to Phytophthora. As a result of this study, genotypes 111-1, 7521, and 1483 were selected for both resistance to root rot and better graft compatibility with chestnut cultivars. Genotypes ‘CHR-1510 , ‘CHR-1370 , ‘CHR-1680 , ‘CHR-1610 , ‘CHR-310 , ‘CHR-1490 , ‘CHR-1470 , ‘CHR-1670 , and ‘7760 also were found promising. Hybridization has been undertaken in several countries, including Portugal, Italy, Australia, and USA, to obtain root rot-resistant root stocks. A limited number of resulting hybrids were used commonly but most of them exhibited graft incompatibility problems. One example is ‘Menzies’ , commonly used as a seedling root stock source in Australia for its resistance to root rot. In Asia, chestnut production is from C. crenata and C. mollissima trees which are naturally resistant to chestnut blight and root rot but are sensitive to the Asian chestnut gall wasp . In Japan, seedlings of ‘Shibaguri’ have been used as scions for production but devastation from the gall wasp has reduced yield. In recent years, due to graft incompatibility problems, they have started using seedlings of the chosen scion cultivar as seedling root stocks. Scions and root stocks can interact at trans-graft-union movement at the molecular level in different ways. In some cases, mobile macromolecules and large signaling molecules can move through the graft union via the vascular system and regulate various physiological processes in scion including vigor, yield, water use efficiency, biotic and abiotic resistance, etc..

RNAs and proteins can be targeted to move up through the graft union and this process has been studied in various vegetable and fruit trees. In addition, some studies have focused on protein production in transgenic root stocks with targeted delivery to scions to control disease. Transgenic root stocks expressing a polygalacturonidase inhibitory protein , were able to protect wild type scion from both a bacterial disease caused by Xylella fastidiosa and a fungal disease caused by Botrytis cinereal, both pathogens use polygalacturonase as a virulence factor. Recently, the strategy of delivering therapeutic proteins from a root stock to a scion was validated in the field where transgenic root stocks were able to transgraft protected a sensitive wild type scion variety from succumbing to Pierces Disease.Transgrafting also holds great promise for the improvement of nut tree root stocks. Commercially accepted scion cultivars grafted onto transgenic root stocks could benefit from the root stock-mediated increase in productivity and/or disease resistance while avoiding potential consumer concerns regarding use of any transgenic scion. Rootstocks can also be improved with enhanced features while simultaneously designed to avoid transmission of macromolecules or products to the scion. As discussed in the biotic stress section, a crown gall resistant root stock was generated by silencing ipt and iaaM genes responsible for tumor formation. Examination for movement from the transgenic root stock to a standard untransformed scion showed that none of the genes or their products transfer through graft union. This method can produce root stocks with enhanced disease resistance or other features while avoiding concerns about changes in the scion or food product.The technique of producing trees on root stocks means two species are genetically joined and therefore can affect one another’s performance. The selection of root stock is an important aspect of orchard management. In nut tree crops, root stocks influence vigor, rooting ability, water and nutrient uptake, bud break timing, yield, nut quality, susceptibility to abiotic factors including temperatures, drought, waterlogging and salinity, and biotic factors, including crown gall, root rot, root-knot nematodes and soil borne fungal infections, harvest efficiency and post harvest nut quality. And now, producing sustainable orchards which can meet the challenges of climate change and economic production, producing better root stocks is even more important. Breeding tree nut crop root stocks began many years ago when local growers near the centers of a species origins started collecting and domesticating the best wild species tolerant to abiotic and biotic stresses and that also produced good nuts.

Advances in irrigation and soil management techniques, along with the application of chemical fertilizers and pesticides introduced by the Green Revolution in farming, resulted in a substantial increase in food production over the last 50 years. However, the potential of chemical fertilizers to disrupt soil health, the food chain, and ultimately human health has led to renewed interest in the consequences of their application, and has resulted in a substantial increase in the number of certified organic farms. Organic systems, defined by management practices lacking the application of synthetic fertilizers and pesticides, appear to reduce the burden of xenobiotics in the food chain, but there is still controversy regarding the nutritional advantages of organic versus conventionally produced food. Irrespective of the impacts on food quality, the potential for organic farming to impact soil health has been expounded as a significant benefit. However, only a few studies have explored these phenomena while taking into account all the confounding variables. Synthetic fertilizers can result in disrupted soil health, and may negatively impact plant growth as well as soil and plant microbial diversity and structure. Therefore, the cumulative use of such compounds could lead to the loss of productivity and economic revenue. Organic fertilizers are known to have several advantages to improve soil fertility, such as the ability to increase organic matter content in soil, improve the soil structure, enhance soil nitrogen content,planting blueberries in a pot enhance nutrient availability, and improve nutrient mobilization as well as increase root growth.

Organic practices rely upon crop rotations, crop residues, animal and/or green manure, off-farm organic wastes, mechanical cultivation, mineral bearing rocks, and aspects of biological pest control to maintain soil productivity and supply plant nutrients. It is generally assumed that greater soil microbial species’ richness promotes enhanced functional stability and thus soil health, but it is unknown whether short-term synthetic fertilizer application versus organic fertilizer will have a substantial impact on these ecological properties. Organic systems have previously been associated with either an increase in soil microbial richness or no significant change. Some of this variance may be explained by differences in the composition of the organic amendment, the time of harvesting, the studied time span, the rotation of crops planted, and so on. However, in general, studies suggest that short-term organic fertilizer amendment leads to a copiotrophic microbial community, while long-term application will result in a more stable community. Overall, Proteobacteria and Firmicutes dominate organic farming systems in long-term field experiments, with plant growth-promoting genera also enriched , while Actinobacteria and Acidobacteria predominate in conventionally managed lands. In the present study, soil type, land use, crop rotation, crop variety, and climate/weather were all standardized in a short-term field experiment conducted on adjacent plots that share the same land use history, same soil edaphic properties, and same environmental stressors . In each plot, a synthetic chemical fertilizer and a natural fertilizer were applied, followed by planting with Solanum lycopersicum and Beta vulgaris.Previously, this system was used to demonstrate that short-term organic fertilizer application did influence the accumulation of essential and non-essential elements within these crops, but overall, there was little or no clear effect of the fertilizer type on the elemental accumulation in the fruits, suggesting that the plant nutritional value was neither improved nor reduced in the short-term.

Here, we employed 16S rRNA amplicon sequencing to characterize how the soil microbial community responded to these treatments and to determine to what extent the community shifts are comparable to the changes occurring in the long term. We identified biomarkers of fertilization type and correlated bacterial shifts with the accumulation of chemical elements in soil and tomato and Swiss chard roots. The proportional differences in predicted bacterial functional genes in response to the fertilizer amendment were also evaluated. Extending the duration of the experiment would help to resolve whether the microbial shifts observed here are persistent over time and determine if the treatments have an impact on soil quality in the long term, which is ultimately required for evaluating the sustainability of land-use regimes. A field experiment was conducted on adjacent plots to study the prokaryotes community diversity and composition of soils under different fertilization treatments, conventional versus organic. In each of the plots, two plant species and B. vulgaris were planted in two subplots . Soils samples were collected over 3 months after fertilization and crop planting.While most studies agree that farming practices impact soil microbiota and the accumulation of elements within the plant, obtaining generalizable conclusions has been difficult, as results are dependent on the applied management, the composition of the organic amendments, the time of harvesting, the time span studied, the rotation of crops planted, and so on.In the present study, which standardizes for differences in soil properties, crop type, and climate conditions, changes in soil microbial richness were observed over the duration of the experiment associated with the crops’ developmental stage. Interestingly, microbial richness was greatest in July and lowest in August for both conventional and organic soils, suggesting that while nutrient supplies start to decrease, there is a decrease in richness.

In addition, the combined effect of ‘sampling date’ and ‘farming system’ significantly correlated with bacterial richness, with the values being higher in organic soils. While the higher richness in organic systems might be in part due to the introduction of microorganisms present in the manure into the soil , Bacteroidetes, and Chloroflflexi,previous data suggest that organic farming systems promote habitat diversification, favoring a more heterogeneous species distribution, or by stimulating the growth of copiotrophic microorganisms.In conventional soils, lower diversity might be expected because of the elimination or growth inhibition of particular bacteria in response to chemical compounds coming from pesticides/fungicides. Predicted functional diversity, however, did not change according to the farming system. pH is known to influence microbial composition as well as the mobility of heavy metals, influencing micronutrients’ uptake. In the present study, while pH was a determinant factor explaining the bacterial community structure found in the soil samples, it did not correlate with the alpha diversity estimate. Despite both plots starting at the same pH values, conventional fertilizer resulted in a reduced pH over time, consistent with the observed impact of chemical fertilizers in longer-term experiments. pH variation influences Proteobacteria, Actinobacteria, and Acidobacteria abundances. For example, Lauber et al. found that the relative abundance of Acidobacteria decreased with pH, while Actinobacteria and Bacteroidetes positively correlated with soil pH. In our study, Bacteroidetes positively correlated with pH,raspberries in pots while Actinobacteria was negatively correlated. The different trends observed in this study might result from the soil having only been fertilized once, which would select for copiotrophic taxa, compared with other studies where multiple recurrent fertilizations were applied over years. In addition, titanium abundance was positively associated with the community structure found in organic soils, and negatively associated with that of conventional soils. In contrast, the abundances of Ag, Mn, As, Fe, Co, Cd, and Ni were mainly associated with conventional soils. Titanium is considered to be a beneficial element for plant growth, improving crop performance through stimulating the activity of certain enzymes, enhancing chlorophyll content and photosynthesis, promoting nutrient uptake, strengthening stress tolerance, and improving crop yield and quality. In agreement, organic soils in the present study shifted towards the enrichment of taxa involved in nutrient cycling as well as in disease suppression. When studying the accumulation of chemical elements within the plants, Liñero et al. documented a differential accumulation according to the fertilization practice. Higher concentrations of Mn, Co, Na, Mg, Cd, and Tl were observed in conventionally grown tomato plants, while Mo, Cu, Zn, K, and Ba abundances were higher in the organically grown ones. Similarly, Swiss chards under organic amendment were more concentrated in Ba and some nutrients. Interestingly, the soil bacterial community of the present study was a good predictor of Mo and Cu accumulation in organic tomato and Swiss chard roots, thus suggesting that those elements’ absorption is favored, in part, by means of the soil microbial activity.

For instance, because of the spraying of copper sulfate on plant aerial parts, a higher accumulation of Cu might be expected in organic roots. Besides, its higher concentration in organic agricultural practices has been previously associated with a higher presence of arbuscular mycorrhizal fungi, and their synergistic interactions with several bacteria, such as species belonging to Rhizobiales and Methylococcales, are already well known. The soil’s bacterial community composition was significantly influenced by the management practice in the 3 months of the experiment, as observed in previous long-term studies. However, the experiment conducted should be extended over time to assess whether the microbial shifts associated with the farming system and the differential uptake of elements by the crops under study persist, in order to evaluate the farming system’s impact on the quality and health of soil, and hence the sustenance of the system. In any case, in the present study, similar to Lupatini et al., but in contrast to Wang et al., organic samples tended to have greater beta diversity compared with conventional samples, suggesting a greater heterogeneity in the microbial composition of organic soils. Hartmann et al. found that ~10% of bacterial and fungal OTUs were specific to the farming system and Lupatini et al. reported that Proteobacteria and Acidobacteria were highly sensitive to the farming practice. In the current study, the abundances of most common bacterial phyla were not statistically different between farming systems, but few phyla were significantly associated with each of the farming systems. For instance, Chloroflflexi, Thermi, Spirochaetes, and Euryarchaeota had greater mean relative abundance in organic soils, while a higher abundance of Firmicutes was observed in conventional soils. Within Chloroflflexi, members of the class Anaerolineae were enriched, which are known for their role in nitrogen cycling and have been previously identified as a highly represented bacterial group in manure. Several other denitrifiers were also augmented compared with conventional soils, including genera Azoarcus and Thauera, Parvibaculum, and Saccharomonospora, while nitrifiers were depleted. Ding et al. observed a similar result when studying microbial community changes in a long-term organic greenhouse farming, where the relative abundances of ammonia oxidizing microorganisms and anaerobic ammonium oxidation bacteria decreased in the organic soil. Furthermore, an increase in methylotrophic bacteria, for instance, those belonging to families Methylophilaceae and Methylococcaceae , as well as the archaea Methanosarcina, was observed, likely associated with their capability to metabolize methane and its derivative compounds that accumulated after the decomposition of the introduced organic matter in organic farming. Taxa belonging to Myxoccocales, Alteromonodaceae, and various Rhizobiales OTUs, known to contribute to general nutrient cycling , were also more abundant in organic soils, similar to previous reports. Members of Ignavibacteria order, suggested to be involved in the degradation of organic matter, were higher in the soil under the organic farming, similar to the results observed in longer-term field and greenhouse studies. However, other groups known to be capable of degrading various complex organic materials coming from manure or compost, such as several genera within Firmicutes phyla, did not respond in that direction. While it could be speculated that they might need recurrent organic amendments to respond, a 12-year greenhouse study suggested that Firmicutes were the least affected phyla by farming system. Interestingly, previous studies report that organic farming systems tend to increase the abundance of microbial taxa associated with plant health promotion. Several members of Firmicutes and Actinobacteria have been associated with disease suppression and have been reported to be augmented in organic farming. However, this was not the case in this study after a one-time manure application, where, for instance, higher abundances of Bacillus, Nocardia, Mycobacterium, and Rhodococcus were observed in the conventional soils. Besides, the plant growth-promoting Myxococcales was ~3.5 times more abundant in organic compared with conventional soils. They are considered to be micropredators that can produce secondary metabolites with antibacterial and anti-fungal activities, killing other microorganisms, and as such, have been suggested to likely play a key role as potential disease-suppressing bacteria in organic farming soils. Interestingly, Myxococcales, unlike the mentioned Firmicutes and Actinobacteria members, have been consistently found to be enriched in organic soils in both short- and long-term experiments, thus suggesting a rapid and lasting response of this bacteria to the organic amendment. Thus, these organisms’ population distribution and functional genes deserve further investigation.

The inoculum of the fungus is highly abundant and ubiquitous and usually comes from infected plant tissues . B. cinerea mainly enters the host via wounds or natural openings . Infections of non-senescing or unripe plant organs usually lead to limited damage and quiescent infections . Different types of quiescence have been described: delay of conidia germination or growth arrest after germination , endophytic symptomless growth in the apoplast , colonization of abscising flower organs followed by growth into ovaries or receptacles where growth arrests . Independent of the type of infection, the pathogen generally enters a short asymptomatic, biotrophic phase at the beginning of the disease cycle . An aggressive necrotrophic phase commonly succeeds the quiescent or asymptomatic phase once plant organs start to senesce or ripen, during which B. cinerea causes rapid decay of the infected tissues . B. cinerea’s infection mechanisms have been studied in model organisms and further characterized thanks to the availability of high-quality reference genome sequences . The fungus is known to actively promote plant susceptibility by employing a variety of virulence factors . In early stages, B. cinerea deploys sRNAs and effector proteins to suppress premature host cell death and immune responses, which enables the fungus to establish inside the host and accumulate biomass prior to the necrotrophic phase . It was demonstrated that B. cinerea Dicer-like proteins DCL1 and DCL2 produce sRNAs that are secreted from fungal hyphae and translocated to the plant cell where they interfere with the host RNAi mechanisms to silence host immune response genes in Arabidopsis and tomato leaves .

Some secreted virulence factors can lead to host cell death,blueberry pot like effector proteins, toxins and enzymes involved in reactive oxygen species production . B. cinerea can also secrete oxalic acid that lowers the pH of the host tissues and stimulates the production and activity of fungal enzymes like pectinases, laccases and proteases . Furthermore, oxalic acid accumulation leads to Ca2+ chelation, which in turn weakens the pectin structures of plant cell walls and inhibits the deposition of callose . Other virulence factors are cell wall degrading enzymes that enable B. cinerea to cause plant cell lysis and loosen walls to facilitate tissue penetration . The fungus is known to produce plant hormones or hormone analogues that may disturb the host’s cellular metabolism and immune responses. The relevance of these mechanisms for the capacity of B. cinerea to infect strawberry remains unknown.Grey mould in strawberries can result from B. cinerea infections of open flowers or by penetration of fruit receptacle tissues . In primary infections, B. cinerea infects flower organs during or right after flowering, allowing hyphae to grow into the receptacle . The sources of primary inoculum range from overwintering sclerotia to conidia or mycelium from infected neighbouring plants . Infected senescent petals, stamens and calyxes can facilitate primary infections in fruit . Histological studies have shown that even though styles are frequently infected, fungal growth appears to be strongly inhibited and never reaches the receptacle. In contrast, fungal growth in colonized stamens can reach the receptacle in some cultivars . Following infection of the unripe receptacle by B. cinerea, fungal growth is usually arrested and a symptomless quiescent phase occurs. The mechanisms that lead to quiescent infections are not yet fully understood. Proanthocyanins appear to induce B. cinerea quiescence in unripe fruit by restricting the activity of fungal enzymes like polygalacturonases that are necessary for aggressive infection of hosts .

Even though PA content in fruit remains constant during ripening, increasing polymerization of PAs leads to lower inhibitory activity in ripe fruit . Similarly, anthocyanins might delay B. cinerea infections or cause quiescence . For instance, strawberries illuminated with white fluorescent light showed increased anthocyanin content and delayed development of grey mould . Reduced fruit decay has also been observed in raspberries with high pigmentation and in transgenic tomatoes that accumulate anthocyanins . Other small phenolics, especially catechins, may have a role in quiescence. High levels of catechins inhibit fungal growth, and a decrease in catechins is correlated with a reduction of other anti-fungal compounds such as lipoxygenases . Interestingly, young and ripe fruit have low catechin concentration, suggesting that initial infections of young receptacles are possible because they do not yet accumulate enough catechins to stop colonization . B. cinerea quiescence is complex and involves additional factors besides the accumulation of phenolic compounds. It has been proposed that quiescence in unripe fruit is initiated by: lack of nutrients such as sugars from the host, presence of preformed anti-fungal compounds, unsuitable environment for fungal virulence factors . In unripe strawberries, factors from all three categories are present, including lack of available sugars , preformed anti-fungal compounds , and high activity of PG-inhibiting proteins . Induction of the necrotrophic phase in ripe strawberries could be triggered by changes in biochemical composition of the host tissues associated with the ripening process, such as increased sugar content, volatile production and alteration of plant deffences . These modifications promote not only fungal growth but also host susceptibility, e.g. via the release of oxalic acid and efflux of toxins .

During secondary infections, the fungus initiates the necrotrophic phase without quiescence . The sources of conidia for secondary infections can also be diverse, from senescent leaves to infected fruit . Conidia from B. cinerea-infected flower parts are major sources of secondary inoculum . It has been estimated that more than 64% of the strawberry infections result from organic fragments that are in contact with the fruit, such as petals and stamens . Contrary to other fruit , senescent flower parts often adhere to strawberries long enough to retain water films for at least 8 h, which is the time needed for B. cinerea conidia germination . Secondary infections can also result from nesting, which corresponds to direct penetration of mycelia growing on neighbouring plant organs such as infected leaves and fruit . Generally, secondary infections proceed rapidly and B. cinerea can complete its germination and infection as fast as 16 h post-inoculation with a rapid increase in fungal biomass at 48 hpi . Early responses of strawberries to infection include higher expression of the defence genes FaPGIP and FaChi 2-1 , whereas lower expression of the reference gene DNA Binding Protein – FaDBP indicates extensive cell death induced by B. cinerea at late stages of infection .Fruit ripening influences the susceptibility of strawberry fruit to B. cinerea . Strawberries are mostly resistant to infection in their unripe stage, where they restrict fungal growth by causing quiescence. However, in the ripe stage, strawberries are highly susceptible and decay rapidly. Fruit susceptibility to fungal disease increases as ripening progresses; hence, B. cinerea appears to promote susceptibility in unripe fruit by activating specific ripening-related processes . In tomato fruit, master transcriptional regulators of ripening have been shown to have different roles in disease susceptibility. For example, the activity of the tomato transcription factor NON-RIPENING favours B. cinerea infection . Strawberries are non-climacteric fruit with a ripening programme different from that of climacteric tomatoes. Thus, a deeper understanding of strawberry ripening regulation and how B. cinerea may modulate particular ripening events are pivotal to characterize the dynamics of the strawberry-B. cinerea pathosystem. Recent transcriptomic studies of developing strawberries point out that ripening events start between the ‘large green’ and ‘white’ stages, and involve changes in cell wall composition, sugar metabolism, hormone biosynthesis and responses, pigmentation and antioxidant levels . Moreover, a general decrease of oxidative phosphorylation processes has been observed during strawberry ripening . Normal strawberry ripening involves a variety of biochemical and physiological processes,nursery pots some of which are discussed below in the context of B. cinerea interactions.Ripening is associated with the disassembly of the fruit cell walls, which leads to tissue softening.

Cell wall degradation benefits B. cinerea as it reduces mechanical barriers to infection and spread, increases the possibilities of bruising and provides the fungus with access to simple sugars as a carbon source . In strawberry, cell wall solubilization occurs early in fruit development when the walls start to swell . Cell wall solubilization correlates with an increase in fruit sugar content, resulting from polysaccharide breakdown. A decrease of acid-soluble pectins and the alcohol-insoluble fraction of cell walls occur during ripening, whereas the water-soluble content increases . The degree of pectin solubilization and depolymerization is highly related to strawberry fruit firmness . Silencing of an endogenous pectin lyase gene in strawberry resulted in fruit with higher external and internal firmness, mostly due to low pectin solubilization, stiffer cell walls, and increased cell to cell adhesion . Besides PL, other enzymes that may have affected strawberry firmness include PGs, β-galactosidases, endoglucanases, α-arabinofuranosidases and β-xylosidases . In addition to the fruit endogenous cell wall disassembly, B. cinerea secretes an extensive array of CWDEs that target most polysaccharides in the fruit cell walls, particularly pectins . These CWDEs include fungal PGs, such as Bcpg2, a gene that is mainly active in the early penetration stage . The expression of B. cinerea PGs is dependent on the host species, the plant tissue, temperature and the stage of infection .Another barrier for B. cinerea infection is the fruit cuticle. During fruit expansion and ripening the cuticle gets thinner, which makes strawberries more susceptible to initial penetration by germinating conidia. B. cinerea can penetrate the plant cuticle by secretion of cutinases . Additionally, cuticle properties can result in higher incidence of cracks and other damages through which B. cinerea can enter the fruit without the need of cutinases . Studies on strawberry cuticles are scarce and only exist for leaf tissues . In tomato fruit, thicker and stiffer cuticles lead to higher resistance to initial B. cinerea infections. Moreover, it is known that the chemical composition of the cuticle changes during tomato ripening, and this is likely to be the case in strawberry .During ripening, the content of sugar in strawberries increases and therefore can serve as nutrients for B. cinerea. In unripe strawberries, the main sugars are glucose and fructose with low concentrations of sucrose. Sucrose levels increase rapidly during de-greening and red colouring . In tomato, it has been shown that the Cnr mutant, which does not accumulate high levels of sugars is still highly susceptible to B. cinerea infection . This observation suggests that even though sugars may serve as a susceptibility factor, high sugar concentrations are not essential for B. cinerea infection. However, sugar content could still influence susceptibility to B. cinerea as specific sugars may serve as ripening initiation signals. For instance, sucrose regulates abscisic acid levels in strawberries, which are necessary for normal ripening and could influence fruit susceptibility as described below . Like other ripening-related events, B. cinerea can alter neutral sugar and sugar acid levels in the infected host tissues, mainly by degradation and depolymerization of cell walls. This was reported for infections in tobacco and Arabidopsis leaves, where the fungus degrades pectins to release the monosaccharide galacturonic acid .ABA biosynthesis during fruit ripening is triggered by a decrease in pH, turgor changes, sugar accumulation, and the switch of sugars from mainly glucose and fructose to sucrose . Effects of ABA on strawberry susceptibility to fungal disease have not been extensively studied, but down regulation of the ABA biosynthetic gene β-glucosidase FaBG3 has been reported to result in fruit with limited ripening and higher B. cinerea resistance . In tomato, ABA accumulation is related to higher pathogen susceptibility, probably via activation of senescence . During strawberry ripening, the increase of ABA is correlated with a decrease of auxin, which induces early fruit growth and expansion but is known to inhibit ripening processes . The role of auxin in fruit susceptibility seems to depend on the plant species, as indole acetic acid treatment in Arabidopsis leads to susceptibility, whereas IAA-treated tomato leaves and eggplant fruit show lower infection severity . Ethylene has a secondary organ-specific role in strawberry ripening, particularly in achenes and green and white receptacles . Ethylene increases the susceptibility of tomato to B. cinerea by inducing ripening; however, its functions during strawberry infections are yet to be fully characterized.

Seeds produced from these crosses were germinated at the University Lindcove Field Station in Tulare County and grown for approximately one year. In 1982, the seedlings were tested by isozyme procedures and the difficult-to-identify nucellars discarded. The hybrid seedlings are being field grown at the Lindcove Field Station and, when they come into fruiting will be tested by Dr. M. L. Roose of the Department of Botany and Plant Sciences for their disease resistance and ultimate horticultural characteristics as root stocks. New possibilities may also arise from recombinations of species, or genera, from graft hybrids or somatic hybrids. Grapefruit develops an excellent root system and although deeply rooted, spreads more widely than that of sweet or sour orange . It is apparently best adapted to growth in heavy or loamy soils and is poorly adapted to light sandy soils. Most Citrus varieties on grapefruit stocks show an overgrowth of the stock at the bud union similar to that of shaddock. In some cases, this is like a shoulder extending out from the bud union, in others it is smooth at the union and flares outward and downward as it approaches the ground line, sometimes referred to as an ‘elephant’s foot’ . The union is, however, cylindrical and shows no tendency for fluting or ridging. The bark is usually slightly thicker than the scion. With some selections, there is a slight bud union overgrowth with lemon scions . At the Citrus Research Center, Riverside, 1927 root stock plantings of trees on grapefruit root developed into normal-sized trees and, at 34 years of age,arandano cultivo were comparable in size to adjacent trees on sweet orange stock and larger than on sour stock with all scion varieties . In spite of their large size, such trees were characterized by surprisingly low yields, which in some cases was only 70 percent of their sweet orange counterpart.

The tendency for low yields has also been noted in Florida by Hume and Ziegler and Wolfe . Fruit size tended to be larger than on sweet orange stock and comparable to that on sour orange or Rough lemon stocks . The larger fruit size might possibly be associated with the fewer numbers of fruit per tree. Fruit quality was good, equal to that on sweet orange stock and frequently slightly superior . Grapefruit root stocks are probably slightly more resistance to Phytophthora than trees on sweet orange or Rough lemon, but are not as resistant as sour orange . That is one of the reasons California growers used it, although Wutscher indicates susceptibility to foot rot was one of the reasons it was discontinued in Florida. California lemon growers also used it because they felt it was more resistant to foot rot than sweet orange or Rough lemon, and that lemon trees budded on it were not as susceptible to shell bark as those on some orange and Rough lemon. Also, phloem necrosis of the lemon scion was less than on sour. They show no resistance to the citrus nematode or the burrowing nematode . Numerous cultivars tested for their resistance to tristeza were variable in their response, some varieties being almost as susceptible as the sour orange, others less, but none were tolerant . Under field conditions, trees declined from natural infection by tristeza at a much slower rate than those on sour orange, erroneously causing some growers in California to assume the combinations were tolerant to the disease. Under California conditions, the stock does not generally stem-pit from tristeza, or only mildly so. In experimental trials conducted by the Citrus Experiment Station, Riverside, the ‘Duncan’ variety did not perform very well. This was the original ‘Duncan’ variety introduced from Florida in the early 1900’s. Recently, all seedy varieties of grapefruit in Florida were lumped as ‘Duncans’. Unfortunately, the original ‘Duncan’ source in the Citrus Variety Collection at Riverside was eliminated and a seedling of it, of doubtful authenticity, substituted.

The original ‘Duncan’ may now be lost. Of all the varieties tested at Riverside, the C.E.S. No. 343 gave the best performance, but even it did not compare with the sweet orange or the sour orange . The C.E.S. No. 343 never gained commercial acceptance. In the eastern Los Angeles basin the ‘Jochimsen’ and the ‘Hall’ were grown. In Ventura County, the ‘Camulos’ was principally grown, and in Santa Barbara County, it was the ‘Stow’. Trees budded on grapefruit stock are less cold resistant in California than those on sour or sweet orange. Although both orange and lemon trees were budded commercially on grapefruit stock, but primarily lemons, results were not outstanding and the stock was never very popular. Because of tristeza, orange trees are no longer propagated on this stock in the U.S. Lemon trees on grapefruit stock can only be purchased through special orders to California nurserymen. Grapefruit cuttings, like shaddock marcots, have performed about equal to the budded trees.The root system, like those of the Valencia and the navel, was less penetrating than those of the seedlings, with a strong shallow lateral system. It is conceivable that in some areas commercial grapefruit orchards under the right conditions could be propagated from cuttings or grown as seedlings and eliminate any bud union problems. In areas where freezes are a major hazard, as in Texas, this could eliminate the practice of mounding to regenerate trees frozen to the bud union or groundline. Several hybrids of the grapefruit have been tried as root stocks and show some interesting results. Among these are the ‘Sampson’ tangelo, ‘Williams’ tangelo, ‘Siamelo’ and the citrumelo. The tangelo will be discussed with the mandarins, and citrumelos with the trifoliate hybrids. The Poorman’s orange is neither an orange nor a grapefruit, although it is used commercially as a grapefruit substitute. Sometimes it is referred to as ‘Morrison’s’ grapefruit or ‘New Zealand’ grapefruit . It is monoembryonic and passes along a genetic scaly bark to its progeny. Like the grapefruit, when budded to Valencia orange it was susceptible to tristeza . The Israeli Poorman is not a Poorman’s orange but something else, perhaps a seedling selection from Poorman .

It is nucellar and does not show the flakey bark of Poorman. It has given fairly promising results in root stock trials in Israel , but its tristeza tolerance is no longer in doubt and it proved susceptible in trials at South Coast Field Station in l97__ . The Natsudaidai of Japan, or Japanese summer orange , is not an orange, or a grapefruit, although it too is commercially used as a grapefruit substitute in Japan where it is the second-most important variety. However, it is used to a very limited extent in Japan as a root stock for the Satsuma. The trees on it are vigorous and do not compare with the more productive trees on trifoliate orange,growing blueberries in containers but the fruit quality is inferior. In California at South Coast Field Station, orange trees budded upon it are tolerant to tristeza, show no stem pitting and are moderately vigorous. However, they are shy bearers . The pummelos or shaddocks, C. grandis [L.] Osbeck , are often confused with the grapefruit, particularly some of the smaller fruited varieties. The pummelos are referred to in some areas as pumplemousse, zabon, buntan, bankan, etc. They differ from most other potential root stocks in one major character, however, in that all varieties of pummelo are monoembryonic and produce no nucellar seedlings. In spite of this, most of their progeny are remarkably uniform in the nursery row and require a minimum of rogueing. When budded to major scion varieties, the scions are remarkably vigorous and uniform in size, even up to an age of 34 years , with surprising uniform performance at the Citrus Experiment Station, Riverside . Some of the better eating commercial varieties like the ‘Alemoen’, ‘Thong Dee’, ‘Kao Phuang’ and ‘Kao Panne’ have been among the most uniform. From the author’s experience, variability is not the major factor in their failure to obtain commercial root stock acceptance . Pummelo fruits, if not parthenocarpic, generally produce an abundance of large seeds, although under certain conditions they may be nearly seedless. Many are self-incompatible and require pollenizers to set seed . The seeds germinate readily and produce vigorous seedlings which are somewhat bushy in character, like the grapefruit, and require considerable training prior to budding. Numerous pummelo varieties have been successfully budded with most of the major commercial citrus varieties in California experiments and observed in various plantings for periods of from 13 to 34 years of age. The reported failure of Satsuma mandarin on pummelo cited by Webber was undoubtedly due to the presence of the tristeza virus in the Satsuma scion. All varieties of pummelos budded with orange scions and inoculated with tristeza by the author , were extremely susceptible to the disease. The reaction of pummelo to tristeza is very similar to sour orange in this respect with many of the young inoculated trees collapsing and dying within a few years. Trees affected with tristeza do not show stem pitting in the stock. This then , is the greatest drawback to the use of pummelo as a root stock, not its variability. In California, especially in root stock trials at the Citrus Experiment Station, oranges, grapefruit and lemons budded on pummelo stocks make large sized trees about equal to those on sweet orange stock. Like grapefruit stocks, yields tend to be somewhat low in proportion to the size of the trees. Like grapefruit, fruit sizes tend to be larger than on stocks like sweet orange and Cleopatra mandarin and more comparable to that on Rough lemon stock . Fruit quality is comparable to that on sweet orange or sour orange stock . The bud union of trees budded on pummelo is good, and like grapefruit stock, shows an overgrowth of the stock.

This may be either a slight shoulder effect or a tendency to slope outward and downward from the union in a slightly pyramidal shape . There is no distinctive character to the bark, except that it tends to be considerably thicker than the scion bark, much thicker than grapefruit. In some instances, the bark may be very thick and extremely rough, like that of an oak tree . Lemon bud union overgrowth does not appear to be a serious problem with the stock in California in the limited plantings observed. The root system of the pummelos is a strong, deeply penetrating tap root system with strong laterals. There are many fibrous roots, with the feeder roots tending to be coarser in texture than feeder roots of most other species . It shows no tolerance to the citrus nematode or the burrowing nematode . Pummelos, however, do have a high resistance to Phytophthora . No problems with gummosis were experienced in any of the author’s plantings. In Thailand, where pummelos are extensively grown, they are propagated principally as marcots and grown on soil beds a few feet above the water table and are relatively short-lived. It is probable that few other stocks would tolerate these conditions as long as the pummelo. In the Philippines, trees grafted on pummelo roots reportedly showed more mottle-leaf disease than on other root stocks observed . In California root stock trials, it was not observed that zinc deficiency was any more of a problem than on the other root stocks. In one lemon root stock trial in Ventura, California, where boron and salt were both somewhat of a problem, the Alemoen shaddock did not show the boron tolerance of C. macrophylla or C. pennivesiculata, but of 40 root stocks, gave essentially the best total performance after 25 years. In Thailand, it is grown under conditions where brackish water would be a problem for many other stocks, but in Thailand salinity, not boron, is the problem. Some varieties of pummelos are not very vigorous when grafted on other root stocks, nor as cuttings, at Riverside. Whether this is due to some unrecognized virus disease, or is of a genetic nature, is not known. Certain hybrids of the pummelo at Riverside have been extremely vigorous, as for example sibs of shaddock X St. Michael sweet orange. One of these hybrids provided fairly interesting root stock results, but proved to be very susceptible to tristeza . The pummelos therefore, have never gained commercial acceptance as root stocks and probably never will in a tristeza area.

The combined top growth of all six root stock seedlings was increased with the addition of VAN fungi and no phosphorus. However, the combined growth with VAN and phosphorus was slightly more. The root stock seedlings decreased in dependency on VAN in the following order: sour orange, Cleopatra mandarin, sweet orange, Rough lemon, Rangpur lime and Carrizo citrange. Root-shoot ratios indicate that root stock mycorrhizal dependency decreases as their capacity for root production increases. Emphasizing the importance of previous work, Timmer and Leyden state the interactions of copper and phosphorus in the fumigation-mycorrhizal syndrome are important. Copper deficiency has been frequently observed in citrus seedlings following the application of phosphate fertilizer. According to Timmer and Leyden, the application of phosphorus induces copper deficiency by stimulating growth of non-mycorrhizal seedlings until copper becomes limiting nutritionally. However, phosphorus-induced copper deficiency appears to be due to phosphorus inhibition of mycorrhizal development of seedlings inoculated with Glomus fasciculatus. Rhodes stresses the fact that mycorrhizae are recognized as being significantly beneficial to host-plant relationships, particularly where root systems are restricted and nutrient systems are low—although he makes no special reference to citrus. Mehraveran also discusses the mycorrhizal dependency of six citrus cultivars .Plants with mycorrhizae and a given phosphorus level are healthier than non-mycorrhizal plants with an equal phosphorus level. The type of mycorrhizae is important; the soil type, area of origin, effects on nematodes, Phytophthora, photosynthetic activity, and hydraulics are all factors to consider. Thus, Menge et al. found that in greenhouse experiments the addition of Glomus fasciculatus significantly increased the growth of Troyer citrange seedlings in 20 of 26 methyl bromide-fumigated soils from Southern California.

Of the six soils in which the mycorrhizal fungus provided no growth increase, two were greenhouse soils ,growing blueberries in pots three were nursery soils, and one was a field soil. Presence of the fungus increased foliar phosphorus, potassium, and copper and decreased foliar magnesium and sodium concentrations in the leaves of the Troyer citrange in the majority of the citrus soils. They present an interesting table on the mycorrhizal dependency of Troyer on Glomus fasciculatus on the 26 soils. While much of the work has been done with sandy soils, the whole soil-mycorrhizal complex is extremely important. Obviously there are different species of the mycorrhizal fungus and one wonders if they are equally effective and if their response varies in a different soil environment. To determine this, Graham, Linderman and Menge tested six VAN isolates. They found that isolates of Glomus fasciculatus was most efficient, and Glomus macrocarpum the least effective. Growth enhancement was significantly greater for Glomus isolates from California than from Florida. It is also stressed that growth enhancement for VAN fungi may vary with the soil type. Johnson reports on the effects of phosphorus nutrition on mycorrhizal colonization, photosynthesis, growth, and nutrient composition in sour orange seedlings. The sour orange seedlings were inoculated with the VAN fungus Glomus intraradices and fertilized with weekly applications of phosphorus. The photosynthetic rates correlated with a high phosphorus content in the leaf tissue of the central plants, but Johnson could find no correlation for the VAN-infected seedlings. He suggests that factors in addition to improved phosphorus nutrition influence the photosynthetic rate of VAN plants. The conclusions of Edriss, Davis and Burger were somewhat similar. Using sour orange seedlings and inoculating them with the mycorrhizal fungus Gigaspora heterogama, they found that the cytokinin production was greater than that of the check non-mycorrhizal plants despite the fact that there were similar dry weights and phosphorus concentrations in the leaves. The enhancement of the cytokinin production seemed to be associated with the mycorrhizal infection rather than increased phosphorus uptake.

The effect of VAN inoculations on nematode populations was first investigated by O’Bannon et al. in greenhouse studies in Florida. They found that when Rough lemon seedlings were inoculated with the citrus nematode, Tylenchulus semipenetrans and then transplanted into soil infected with VAN Glomus mosseae, that the presence of the fungus increased seedling growth. The seedling suppression by the citrus nematode alone was greater than the checks or the VAN inoculated. They made no studies on other nematodes such as the burrowing nematode Radopholus similis. Similarly, Hussey and Roncadori report that nematode suppression of vegetative growth or yield are partly offset by the presence of a VAN fungus. Using Rough lemon seedlings they found that the presence of the mycorrhizal fungus lessened the nematodes’ attraction to the citrus roots, hindered penetration, and the subsequent development and reproduction of the citrus nematode was suppressed. They do not specify why, except that a healthier plant is a more resistant plant. To some extent, this same hypothesis exists with the VAN fungus relationship with Phytophthora. Davis and Menge working with Pineapple sweet orange seedlings, present evidence that suggested that there was some tolerance to Phytophthora parasitica in seedlings infected with Glomus fasciculatus. They also felt this effect was caused by the ability of mycorrhizal roots to absorb more phosphorus and possibly other nutrients than non-mycorrhizal roots, as evidenced by root health and greater phosphorus uptake. Again, a healthier plant is a more resistant plant. They did not report on any studies with Phytophthora citrophthora. Davis and Menge again suggest that VAN fungi have a variable influence on the tolerance of seedlings of Pineapple sweet orange and Troyer citrange to Phytophthora parasitica. There are a number of papers on the effects of VAN fungi and water relationships of host citrus seedlings. The first report perhaps was that of Levy and Krikun . These two Israeli researchers working with Rough lemon seedlings studied recovery from water stress on similar-sized VAN infected seedlings and non-mycorrhizal seedlings. They found the VAN-infected seedlings affected stomatal conductance, photosynthesis and proline accumulation but not leaf water potential.

They suggest that most of the effect of mycorrhizal association is on stomatal regulation rather than on root resistance. Syvertsen studied the hydraulic conductivity of four commercial citrus root stocks. The hydraulic conductivity was estimated using a special pressure chamber technique. He found that Carrizo citrange and Rough lemon had the highest root conductivity, whereas Cleopatra mandarin and sour orange had the least. Further hydraulic studies were conducted by Graham and Syvertsen . They used seedlings of Carrizo citrange and sour orange grown in a low phosphorus sandy soil and either inoculated with Glomus intraradices or fertilized with phosphorus. The mycorrhizal-infected seedlings had sufficient levels of leaf phosphorus, but the non-mycorrhizal seedlings were phosphorus deficient. The root-shoot ratio of both root stocks was reduced by the mycorrhizal colonization, but root hydraulic conductivity per unit root length of mycorrhizal Carrizo and sour orange was more than twice that of non-mycorrhizal seedlings. The mycorrhizal plants had higher transpiration rates, apparently increased bythe conductivity of the roots. The authors felt the response was due to the mycorrhizal enhancement of phosphorus nutrition. In further studies, Graham and Syvertsen grew seedlings of five citrus root stocks in a low phosphorus sandy soil. The seedlings were incorporated into three treatments: inoculated with Glomus intraradices, non-inoculated, but fertilized with phosphorus, and non-inoculated and no phosphorus added. The order of the mycorrhizal dependency of the five root-stocks is as follows: sour orange = Cleopatra mandarin > Swingle citrumelo > Carrizo citrange > trifoliate orange. The less dependent root stocks, i.e., trifoliate orange and Carrizo citrange, had greater leaf phosphorus, finer roots, and slower growth rates than sour orange and Cleopatra mandarin. Rootstocks with a lower mycorrhizal dependency also generally had greater hydraulic conductivity of the roots,frambuesa cultivo greater transpiration and carbon dioxide assimilation rates. In additional but similar studies, Syvertsen and Graham amplify their work again with seedlings of Carrizo citrange, trifoliate orange, sour orange, Swingle citrumelo, and Cleopatra mandarin. Whole plant transpiration and maximum rates of net gas exchange or carbon dioxide and water vapor from single leaves were positively correlated with the hydraulic conductivity of the seedlings. Leaf nitrogen and phosphorus content and shoot-root ratio were also positively correlated with root conductivity. The differences in soil water depletion and plant water relations of trifoliate orange and Carrizo citrange during drought and recovery cycle is related to their root conductivity. They stress that the capability of root systems to conduct water and mineral elements is a very important factor in plant growth and physiological activity. The possibilities of seed inoculation or field inoculation was first mentioned by Newcomb . Hattingh and Gerdemann inoculated sour orange seed, successfully developing a special technique. They coated the seed with a mycorrhizal inoculum in a 1% solution of methyl cellulose. Menge, Lembright and Johnson indicated that commercial production of mycorrhizal inoculum for use in fumigated or sterilized soil was being attempted in several locations in the United States.

They felt the only current way to produce suitable quantities of mycorrhizal inoculum was on roots of susceptible host plants. Contamination by other pathogenic organisms can be a problem. Maybe better methods are available. The most common method for inoculating citrus in the field and in the greenhouse has been to mix the mycorrhizal inoculum with the soil prior to planting or transplanting. Menge et al. felt banding, layering and root inoculation were more efficient than seed inoculation. Root fragments can also be an important source of inoculation. Graham and Fardelmann found that root pieces stored up to one year under moist conditions did not lose the colonization potential with Glomus epigaeum. However, drying reduced this potential to nearly zero after nine months. Glomus intraradices was found sporulating in citrus roots found in orchard soil. They propose that dead root fragments account for a high percentage of the propagules in the citrus soil. The propagation of mycorrhizae cannot be too difficult. For a number of years a citrus nurseryman in California offered mycorrhizae for sale for inoculative purposes. This service is no longer available either because of propagation problems or little demand by the citrus industry. The knowledge of the necessity of the mycorrhizae in the seed bed or the transplant stage is the important thing. The mycorrhizae will gradually re-infect a fumigated soil. The nurseryman could leave the fumigated site fallow for a year or plant host cover crops of cereals and grasses or legumes prior to planting a seedbed or transplanting. Furthermore, field grown nursery trees are gradually diminishing. Container growing is so much more efficient and economical, and container trees can be grown in a shorter period of time. There is also less transplant shock. When the author visited South Africa in 1982, there were only three field grown nurseries left and they ceased with that planting. South Africa is essentially 100% in container growing. Spain and Australia are now heavily into container growing, with more citrus producing areas following suit. If the container planting mix is properly planned and prepared, there will no longer be this problem. For further information on this problem, a nice review of the total VAN potential benefits and interactions is presented by Graham . While the article is a review, it nicely presents the views and facts in pathology, horticulture, and physiology. A book on the subject is currently being written by J. A. Menge of the Department of Plant Pathology, Citrus Research Center, Riverside. Differences in cellular structure have infrequently but effectively been used to distinguish a limited number of stock species. Thus, Penzig found a striking difference in the cellular structure of the pith of twigs of trifoliate orange and sour orange, and this may probably also be true of the roots. Swingle suggested this method of distinguishing between these two species when used in Satsuma production in the U.S. Gulf states. Longitudinal sections of the pith of young stems of the trifoliate orange exhibit an irregular arrangement of thin-walled cells [labeled as] Fig. 66A, while similar sections of sour orange stems show only uniform thin-walled cells arranged in regular series and an entire absence of the crossplates of thick-walled cells . Wolf extended this method to distinguish Yuzu, which was found to have only thin-walled pith cells similar to those of the sour orange, but irregularly arranged . Thus, it differs from the sour orange, in which the cells are arranged in regular series or chains, and from the trifoliate, in which there are crossplates of thick-walled cells .

This antinociceptive response, termed stress-induced analgesia , is mediated, in part, by the release of opioid peptides. However, opioid-dependent and opioid-independent forms of SIA can be differentially activated based upon stressor parameters and duration. Recent research in our laboratories has demonstrated that an endocannabinoid signaling system mediates nonopioid SIA induced by continuous foot shock. A role for cannabinoid CB1 receptors in SIA was demonstrated by our observations that competitive CB1 antagonists, administered systemically or locally in the dorsolateral periaqueduc-tal gray , block nonopioid SIA. Furthermore, SIA is attenuated in rats rendered tolerant to cannabinoids, but not in rats rendered tolerant to morphine. In the midbrain PAG, a key structure implicated in the descending control of pain, stress triggers the rapid mobilization of two endocannabinoid lipids—2-arachidonoyl glycerol and anandamide. These compounds are hydrolyzed in vivo by distinct serine hydrolases. Anandamide is degraded by fatty-acid amide hydrolase, whereas 2-AG is hydrolyzed by monoacylglycerol lipase. Inhibition of either FAAH or MGL in the PAG also enhances SIA in a CB1-dependent manner, further supporting a role for endocannabinoids in regulating expression of SIA at the supraspinal level. The distribution of CB1 receptors in the brain suggests several anatomical regions where endocannabinoid actions could modulate SIA. One such region is the amygdala,best indoor vertical garden system an area of the limbic forebrain implicated in both fear conditioning and affective dimensions of pain. An ascending spino-pontoamygdaloid circuit has been postulated as an “affective” nociceptive pathway. CB1 immunore activity is dense in the basolateral nucleus of the amygdala, but is reportedly absent in the central nucleus of the amygdala. CB1 immunore activity is associated with a distinct sub-population of GABAergic interneurons in the BLA, corresponding to large cholecystokinin-positive cells.

The distribution of FAAH and MGL at this site also correlates well with the distribution of CB1 receptors. The anatomical localization of CB1 in the BLA is consistent with electrophysiological data demonstrating that activation of these receptors presynaptically modulates GABAergic transmission.For example, a form of short-term synaptic plasticity – depolarization-induced suppression of inhibition – in the BLA is blocked by CB1 antagonists. BLA efferents innervate the CeA, the main amygdaloid output nucleus, which sends projections to the PAG and other regions. Thus, an endocannabinoid-mediated reduction of GABA release would disinhibit principal neurons innervating the CeA, to control information processing in the amygdala. Unilateral micro-injection of cannabinoid agonists into the amygdala also induces antinociception in the tail- flick test, supporting a role for this structure in modulation of pain sensitivity. Furthermore, micro-injections of the GABA agonist muscimol in the CeA attenuates cannabinoid antinociception. Unilateral or bilateral lesions of the CeA also suppress the antinociceptive effects elicited by both systemic cannabinoids and diverse environmental challenges. Endocannabinoid signaling in the BLA also mediates extinction of aversive memories, suggesting that endocannabinoids modulate multiple responses to stress via actions in the amygdala. In the present study, we investigated the role of cannabinoid CB1 receptors in the BLA and CeA in nonopioid SIA in rats. First, the CB1-selective antagonist/inverse agonist rimonabant was micro-injected into the BLA and the CeA to examine the contribution of these sites to endocannabinoid-mediated SIA. Based upon the distributions of CB1 receptors in these sites, we hypothesized that pharmacological blockade of CB1 receptors in the BLA, but not the CeA, would suppress nonopioid SIA. To examine the contribution of endocannabinoids in the amygdala to SIA, we administered selective pharmacological inhibitors of FAAH and MGL locally in the BLA, at doses that enhanced nonopioid SIA following micro-injection into the midbrain PAG.

To this end, we used two pharmacological inhibitors that selectively target either FAAH or MGL. The FAAH inhibitor URB597 increases brain accumulation of anandamide but not 2- AG and enhances SIA in a CB1-dependent manner. On the other hand, the MGL inhibitor URB602 increases levels of 2-AG, but not anandamide, in the midbrain PAG and enhances CB1-mediated SIA when micro-injected into this structure. Sixty-three male Sprague–Dawley rats were used in these experiments. All procedures were approved by the University of Georgia Animal Care and Use Committee, and followed the guidelines of the International Association of the Study of Pain and the National Institutes of Health. Rimonabant was obtained from NIDA. URB597 was purchased from Cayman . URB602 was synthesized by reacting diimidazole- 1-ylmethanone with biphenyl-3-yl amine in acetonitrile in the presence of 4-dimethylaminopyradine and subsequently with cyclohexanol as described previously. Animals were anesthetized with a mixture of sodium pentobarbital and ketamine. Stainless steel guide cannulae were unilaterally implanted above either the BLA or CeA using zero points from bregma, the midline suture and the surface of the skull, respectively. Cannulae were fixed to the skull using skull screws and dental acrylic. Five to seven days after surgery, rats were habituated to restraining tubes prior to testing. The latency to remove the tail from a radiant heat source was measured using the tail-flick test. Drug or vehicle was micro-injected using a micro-infusion pump over 60 s into either the BLA, CeA or deliberately off-site. In experiment 1, rimonabant or vehicle was micro-injected into either the BLA or CeA 5 min prior to foot shock. In experiment 2, URB597 , URB602 , or vehicle was administered to the BLA 32 min prior to foot shock. Doses and delays were selected based upon previous studies demonstrating efficacy of the identical drug treatments following micro-injection into the midbrain PAG. SIA was induced by exposing rats to continuous foot shock using a Lafayette grid-shock apparatus and quantified behaviorally using the radiant heat tail-flick test. Removal of the tail from the heat source terminated application of thermal stimulation. Tail- flick latencies were monitored over 4 min immediately prior to exposure to the stressor to evaluate changes in basal nociceptive thresholds induced by pharmacological manipulations.

Tail withdrawal latencies were measured at 2-min intervals before and after foot shock. A ceiling tail-flick latency of 10 s was employed to prevent tissue damage. In all studies, the experimenter was blinded to the experimental condition. Following testing, rats were euthanized with sodium pentobarbital and perfused with saline followed by formalin. Brains were removed, cryoprotected overnight, cryostat-cut , and mounted onto gelatin-subbed slides. Sections were dried and stained with cresyl violet. Injection sites were confirmed histologically using a light microscope. micro-injection sites were confirmed for thirty-seven animals in the BLA and fifteen animals in the CeA . Eleven animals were used as off-site controls. Only animals with histologically confirmed micro-injection sites were included in data for analysis. Tail flick data were blocked for each subject by averaging every two adjacent tail-flick latencies into a single mean, as described previously. Means of two-trial blocks, calculated for each subject, were subjected to repeated measures analysis of variance and ANOVA, as appropriate. Post hoc comparisons were performed using the Fisher’s protected least squares difference , with P < 0.05 considered significant.In all studies, baseline tail-flick latencies did not differ between groups prior to administration of drug or vehicle. Moreover, latencies recorded just prior to foot shock, following injection of drug or vehicle, were similar between groups, indicating that the injection alone was not sufficient to induce antinociception. In all studies,plant pot with drainage foot shock increased tail-flick latencies .By contrast, intra-CeA micro-injection of rimonabant did not alter tail-flick latencies relative to vehicle . Neither the FAAH inhibitor URB597 nor the MGL inhibitor URB602 altered SIA , at doses that markedly enhanced SIA following micro-injection into the midbrain PAG. The present study demonstrates that pharmacological blockade of CB1 receptors in the BLA attenuates nonopioid SIA induced by continuous foot shock. micro-injection of rimonabant into the BLA, where CB1 receptors are dense, suppressed stress-induced antinociception relative to control conditions. By contrast, micro-injection of rimonabant into the CeA, where CB1 receptors are largely absent, failed to suppress nonopioid SIA. micro-injection of rimonabant into regions outside the amygdala also failed to suppress SIA, suggesting that the actions of the cannabinoid antagonist were not due to diffusion to distal sites. Our data are consistent with the observation that CB1 agonists depress monosynaptic evoked inhibitory post-synaptic potentials in the BLA but not in the CeA. Our results, therefore, suggest that CB1 receptors in the BLA modulate local inhibitory networks in the BLA to ultimately regulate expression of SIA. Nonetheless, neither the FAAH inhibitor URB597 nor the MGL inhibitor URB602 enhanced SIA following micro-injection into the BLA, at doses that markedly potentiated SIA following micro-injection into the midbrain dPAG. These differences likely reflect differential modulatory roles of distinct endocannabinoids in the ascending “affective” pain pathway compared to descending pain modulatory systems. Anatomical studies suggest that CB1 is preferentially associated with GABAergic, as opposed to glutamatergic, synapses in the BLA . Nonetheless, in the lateral amygdala, endocannabinoids mediate reductions in both local inhibitory inputs as well as excitatory transmission, whose actions could exert opposing effects. Low frequency stimulation of the lateral amygdala also mobilizes endocannabinoids from BLA neurons to presynaptically induce a long-term depression of inhibitory GABAergic transmission. Endocannabinoid LTDi in the BLA in turn, enhances excitatory synaptic transmission in the CeA. A specific role for anandamide, but not 2-AG, in this form of neuronal plasticity is suggested by two complementary observations. First, LTDi is enhanced in FAAH−/− mice, which are impaired in their ability to metabolize anandamide. Second, endocannabinoid mobilization in LTDi apparently requires the activation of the adenylyl-cyclase-protein kinase A pathway in the BLA, but not the phospholipase C-diacylglycerol lipase pathway that is implicated in 2-AG formation.This latter finding is in contrast to our observations that 2-AG appears to be the primary endocannabinoid implicated in nonopioid SIA; a strong temporal correspondence exists between cannabinoid SIA and the accumulation of 2-AG, but not anandamide, in the PAG.

Liquid chromatography mass spectrometric studies are required to determine how foot shock-induced endocannabinoid mobilization in the BLA differs from that observed previously in the PAG. It is also possible that changes in SIA could be more prominent when supraspinally-mediated measures of antinociception are employed that are more sensitive to the affective dimensions of pain . Our data do not preclude the possibility that the doses of FAAH and MGL inhibitors employed here were unable to surmount high levels of FAAH and MGL activity at this site relative to the PAG. It is also possible that enzymes other than FAAH or MGL may participate in endocannabinoid deactivation at these sites. We propose that CB1 receptor activation in the BLA removes inhibitory control over projection neurons innervating the CeA. The CeA thus receives multi-modal sensory information, and in turn, coordinates appropriate behavioral, hormonal, and autonomic responses to stress via efferent projections. The lack of effect of the FAAH inhibitor in our study is consistent with previous work demonstrating that FAAH−/− mice and mice treated with URB597, administered systemically, show similar amygdalar activation in response to restraint stress compared to control mice. In the BLA, restraint stress produced a low level of Fos induction, which was unaffected by cannabinoid treatment, whereas, the combination of restraint stress and CB1 agonist administration produced robust Fos induction within the CeA. These data support a synergistic interaction between environmental stress and CB1 receptor activation in the amygdala that could contribute to the behavioral phenotype observed here. CB1 as well as FAAH and MGL immunoreactivity are abundant in the BLA, suggesting that incoming stimuli may trigger the on-demand formation of endocannabinoids to activate CB1 receptors prior to undergoing enzymatic hydrolysis. Activation of CB1 receptors in the BLA, in turn, reduce the inhibitory tone exerted on principal neurons, eventually propagating this signal to brain regions implicated in antinociception, most notably the PAG. In the dPAG, foot shock stress stimulates mobilization of the endocannabinoids, 2-AG and anandamide, and micro-injection of CB1 antagonists into this site virtually abolishes nonopioid SIA. Our results suggest that endocannabinoids may be differentially modulated by stress in the amygdala. For example, in the amygdala, anandamide levels are decreased after the first exposure to restraint stress whereas 2-AG levels are unchanged. Our results collectively suggest that the facilitatory effects of endocannabinoids in specifically enhancing antinociceptive responses to foot shock stress occur downstream of receptor interactions in the BLA.In a variety of ways, Colorado remains a state in transition. As is the case with many states around the nation, the economy continues to improve from the recession. Demographically, the state is becoming increasingly diverse.

Root growth is cyclic, it generally alternates with shoot growth, usually precedes it, and synchronizes with trunk growth. In Israel, Cossman reports growing roots could be found throughout the entire winter season. The most intense root growth periods were in the spring and autumn and of relatively short duration.Monselise also states that root growth alternates in most cases with shoot growth and stresses that soil temperature and soil moisture are important factors. In California, Crider observed that roots do not grow continuously but alternate with shoot growth. Total root elongation and greatest rate of growth was much greater during the first rather than the second and last periods . Total root growth as we1l as the rate of growth during the second or mid-season growth stage was decidedly less than the first and last period. The rate of root elongation was considerably greater during the night rather than during the day. Roots were inactive during the winter and early spring, and Crider suggests that this is the best time to cover crop or interplant. Waynick and Walker observed in California that growth of the roots in the subsoil preceded growth in the upper foot of soil by nearly 28 days in the spring. Root growth preceded shoot growth except in the fall, when the growth cycle seemed to coincide. This condition may vary in other citrus areas. They also noted a difference in root growth with different scions and different aged trees. In October, when the last growth cycle was completed, there was only scattered growth of roots in the upper 45 cm of soil with old trees. Grapefruit Trees showed little development while Valencia trees showed a moderate growth,hydroponic vertical garden but only in the upper 56 cm of soil. In contrast, yearling Valencia trees in the same soil developed a heavy root growth.

Waynick and Walker measured soil temperatures at 15-35 cm, 51 cm, and 66 cm. The soil temperatures at 36 cm and above were consistently lower by 2°C than lower depths until mid-April. The average subsoil temperature in [“Check” appear here in typescript in the margin of the manuscript] late March was 13.8°C, or just above the minimum of 12°C reported by Girton . The soil temperature below 51 cm averaged 1.5°C less than that from 36- 45 cm, and it was in this area that the most active root growth took place. In March, subsoil root growth was first observed. There was no root elongation above 36 cm, but root growth was general between 36-51 cm. The growth rate of the roots varied from .62—1.60 cm daily. Rootlets elongate at the rate of .32-1.27cm per day in an active cycle. Root growth ceased the first week in April, when top growth occurred. Root growth resumed in September and lasted five weeks. In a three week period the rootlets grew as much as 36 cm, and the size of the roots doubled. Their data show growth of the roots is cyclic and precedes top growth. There is a brief rest period when roots cease to elongate and the growth of the top begins. There are 2-3 month periods when the roots fail to elongate. Roots make very poor growth when severed by cultural equipment, even in two year’s time. They emphasize the important relationship of irrigation and supply of nutrient to the growth cycle of the roots, and question the possible undesirable effects of the heavy application of cold irrigation water when the root cycle is just starting. In Florida, Ford states that citrus root concentration fluctuates extensively during the year. It had been assumed that roots grow in alternate cycles to growth flushes, but this did not seem to apply to the situation in Florida. Root concentration was usually lowest in August and highest in September. He stresses the importance of taking root samples at the same time of year. Soil temperatures in Florida citrus orchards are higher than those in California throughout the root zone during the entire year . Tully also noted that in citrus trees shoot and root growth usually alternate. Root systems are generally observed and classified by either complete excavation of the root system, digging trenches, push and pull procedures, removal of soil cores, or use of special technical devices. Factors affecting root distribution are the scion variety, environment, temperature, age of tree, water relations, drainage, salinity, nutrition, diseases, soil types, soil stratification, pruning, cover crop, etc.

Therefore, it is understandable that there is considerable discrepancy in the descriptions of root systems as described by various observers. An old adage is that lateral roots of a tree extend radially from the trunk to a distance equal to that of the height of the tree. A 4.6 meter tall tree would thus have roots extending laterally in all directions to a distance of 4.6 meters. There are, of course, many exceptions to this rule as root distribution is affected by many factors. Under many circumstances, the lateral spread of the roots is such that at any normal orchard spacing there is considerable intermingling of roots from adjacent orchard trees. Perhaps the earliest recorded observations on citrus root systems are those of Mills in California. On excavated trees grown on [“Check” appear here in typescript in the margin of the manuscript] loam soils in Ventura County, he characterizes sweet orange as having a shallow root system. The main mass of the root system of bearing trees on sweet orange stock are concentrated in a horizontal layer about 45 cm thick, the top of which was 20-25 cm from the surface, with crooked tap roots. The tap root of grapefruit was also crooked. The lateral roots develop at the expense of the tap roots, making a prodigious root system. A seven-year-old seedling had a tap root 120 cm long. The largest lateral root started 60 cm below the soil surface and extended for a distance of 8 meters at an average depth of 45 cm below the surface. Over 90 percent of the root system was confined to a layer 25-60 cm below the surface. Grapefruit had a much larger number of fibrous roots than sweet orange, and the roots were deeper beneath the surface, the majority below 38 cm. A nine-year-old navel tree on sour orange had the largest and uppermost lateral, which started 15 cm below the surface and descended immediately at an angle of 45° to 2.5 cm below the surface to a distance 3 M from the tree, when it rose to within 20 cm of the surface. It was then cut by cultivation equipment, grew downward 2.5 cm and horizontally 2.4 meters more. The longest tap root was approximately 3 meters, maybe the deepest recorded in California. Those trees on sour orange with few laterals were less productive than those which had numerous laterals near the surface. Hume states that in Florida’s Hammock soils, sour orange roots are produced abundantly and penetrate well into the soil. He felt that this was an advantage over sweet orange since the trees were not so readily affected by variations in soil moisture. The roots penetrate sufficiently deep to be more or less in contact with a permanent water supply.

Hume also says that under these conditions, sour orange suffers less under prolonged drought under identical conditions than Rough lemon. With Rough lemon the main roots show a wide variation. In all cases the crown-roots extend a considerable distance from the trunk and a strong tap root is produced, but occasionally the large lateral roots lie close to the surface and most of the feeder roots are in the top 38 cm of soil. Hume also states that the root system produced by trifoliate orange is very good,vertical vegetable tower as the roots penetrate well and fibrous roots are produced in abundance. He also cites the California experience of Mills that sweet orange is not deep rooted and that most of the roots are in the top 46 cm of soil. There is lack of evidence in Florida, but in nursery trees, [“Check” appear here in typescript in the margin of the manuscript] root development of trifoliate orange was equal to sour orange. The root system of grapefruit was well developed. In Egypt, El Sawy found the root system of lime heavily branched with abundant fibrous roots. Those trees on citron, sweet lime, and sour orange were sparsely branched with less fibrous roots. The depth of penetration of citron or sweet lime was 41-46 cm, 61-71 cm on lime, and 112-168 cm on sour orange. As far as lateral extension, the citron had the least, followed by sweet lime and those on sour orange. Brown and El Sawy observed in Egyptian orchards that the sour orange adapted to heavy soils and was not readily affected by variations in soil moisture. It provided good root anchorage, roots in loamy soils penetrating to a depth of 110-170 cm with a lateral spread of 4-10 M depending on the scion variety. They made no comment about the root system of sweet orange. The grapefruit and shaddock they felt were well adapted to heavy and moist soils, with their root systems well developed and evenly distributed. The Baladi lime was adapted to sandy soils, but not wet or heavy soils. The root system was richly branched and had more fibrous roots than any of the other stocks. The bulk of the roots in sandy soils penetrated an average depth of 70 cm and occasionally to 250 cm. Lateral roots extended to 7 M. The Rough lemon was adapted to sandy soils where sour orange failed to produce a satisfactory tree. Although it had a strong tap root which penetrated deeply into the soil, there were abundant lateral roots which were well and widely distributed and were nearer to the surface than sour orange. The Rough lemon had many feeder roots. The sweet lemon was also not recommended for heavy soils. Its root system was characterized by downward penetration and lateral expansion similar to that of the Baladi lime, but was sparsely branched and had less fibrous roots in the upper 30 cm of soil. There were no comments on the citron root system other than that it did very poorly on sandy soils. In Israel, Oppenheimer examined the root systems of 19-month-old trees on sweet lime, sour orange, Rough lemon, citron, Baladi , Shamouti, sour orange, pummelo, and grapefruit. He says all Citrus root systems belong to the intensive type. The lemon-lime group are characterized by strong root branches whose horizontal spread does not fall short of their penetration in depth, and with a multitude of fine absorbing rootlets. Single tap roots are rarely found after transplanting. The surface layers of soil are well provided with roots, except the root system of sour lemon, which showed rather fine root branches and a tendency for deep penetration without strong crown root formation in the surface layers. With Rough lemon the lateral roots changed direction from the horizontal and turn downward. He agreed with Hume in praise of Rough lemon, “because of the great foraging power of its roots.” With sweet orange, he observed a tendency to form a strong tap root system which showed good ramification and a considerable development of horizontally growing main roots. He did not agree with observations by Mills in California on this, but did agree with Hume’s Florida observations, perhaps because of soil differences. Hume, in Florida, worked in super drained, very sandy soils, warm enough throughout the year for good root growth and root activity. The sour orange, grapefruit, and pummelo tend to form root systems of a conical shape with well developed penetrating central roots, and a monopodial character. None of the upper roots are strong, and most of the lateral roots descend obliquely. The abundance of fibrous roots on grapefruit produces more fibrous roots than sweet orange or sour orange. In Florida’s deep sandy soils, Savage, Cooper, and Piper examined sixyear-old trees of Parson Brown on 15 root stocks planted at a distance of 30-92 cm. However, some roots were lost in the excavation. Sour orange showed three or more well developed tap roots penetrating 90-125 cm deep. There were numerous small laterals in the upper foot and some of the laterals extended 150 cm from the trunk.

There are many similar top working examples too numerous to mention. Eliminating the lemon trunk, which was usually so susceptible to shellbark, appeared to have the same effect as high budding and eliminated the shellbark. Trees affected by known diseases probably under no circumstances should be top worked. In some instances top working may result in an incompatibility. For example, in 1976 while visiting Japan, the author was shown some satsuma trees on trifoliate orange root stock which had been top worked to Uwa pummelo. An incompatibility occurred at the juncture of the Uwa pummelo and the satsuma. In other instances, the top working may result in the susceptibility to a disease which would not have affected the original combination prior to the top working. To illustrate the point, lemon trees on sweet orange root stock are not susceptible to tristeza. However, if the combination was to be top worked to sweet orange the new combination would be susceptible to tristeza . Trees showing symptoms of tristeza should not be top worked to lemons; but susceptible trees can be top worked successfully if done before tristeza occurs . Growers in Spain, until the occurrence of tristeza, probably did more top working than in any other citrus area of the world. Citrus diseases are easily spread through top working, either by the mechanical means of contaminated tools, or the use of scion varieties in which symptoms of the presence of a pathogen were not expressed. In 1983, Dr. Luis Navaro, Director of INIA at Moncada , Spain,vertical hydroponics told a group of participants at the First International Society of Citrus Nurserymen that of all the Spanish citrus cultivars entered into the new budwood certification program established in Spain, over 50 per cent of the cultivars were carrying three or more viruses.

To make this information more available, Platt prepared a handout on citrus top working compatibility. It is reproduced intact in Table 12. While Platt prepared this information specifically for California growers, citrus growers in other areas will find that many of the recommendations can be appropriately applied to their orchard conditions as well. The largest related incidence of top working in the California citrus industry occurred in the early 1960’s. The incident had nothing to do with tristeza directly, but was associated with the rapid increase in citrus plantings in the 1950’s, particularly in the San Joaquin Valley, following the destruction of orchards in the Los Angeles basin by tristeza. The causal factor was a citrus bud variation referred to as the “acid-navel.” . This was a mutant of the ‘Frost’ navel which was so extensively propagated at that time. The variant was a fruit mutation characterized by smaller fruit, coarser rind, and other undesirable external characters. However, the interior of the fruit had a more watery-appearing flesh with juice of a higher than normal citrus acid content; hence the term “acid-navel.” The fruit characteristics were completely unacceptable for commercial purposes. Since the trees were disease free and no incompatibilities were involved, most of these trees were top worked, generally to another navel orange of the grower’s choice. Hundreds of acres, literally thousands of trees, were top worked. The episode stresses the importance of nurserymen cutting buds for propagation from fruiting trees so that the fruit characteristics of the scion variety can be observed. Inter stocking of citrus trees has not always proved useful. Diseased trees should not be top worked, and when and if top working is necessary, it should be with full knowledge of the consequences involved.

The fact that citrus inter stocks, except for preventing certain incompatibilities, have had so little effect on the physiological response of the scion should not imply that they may never be successful. Most citrus inter stocks have been genetically close and oftentimes commercial varieties. Maybe there are non-commercial citrus cultivars or citrus relatives which may produce desirable effects. The success of inter stocks with a tree crop like apples cannot be overlooked. Coincident with any discussion of inter stocks are other horticultural procedures, which may not be so difficult to perform, but perhaps could produce effects similar to inter stocks, and actually be considered as modified inter stocks. The first of these procedures is the ring graft. Considerable interest was generated in the 1930’s through the 1950’s in the technique . Much of the work was done with apples and other tree crops. The procedure is accomplished by using knives such as linoleum knives or grape knives with a curved point at the top of the blade. The knives may have spacers between the blades or handles so that parallel cuts can be made around the trunk or branches of the tree. Considerable pressure is put on the cutting implement so the blades penetrate through the phloem . It is impossible to control the pressure and, as a result, the knife blades cut not only through the phloem but also through the cambium and into the xylem elements. Penetration into the xylem may easily range from 1/32 to 1/8 inches . Thus, not only are the phloem elements severed, but xylem elements also, resulting in a xylem block as well as a phloem block. A single cut through the bark of the tree trunk is referred to as girdling or ringing . The technique is used almost exclusively to increase fruit set in navel oranges, although it is occasionally practiced with some mandarin varieties. The cut is not protected in any manner from desiccation. The resulting severance of both the phloem, cambium, and xylem tissues apparently heals quickly. Furr, Reece, and Hrnciar found there is a return to normal nutrient absorption two weeks after girdling, which suggests that the break in the cut tissues may be at least partially healed over in two weeks.

Curtis and Clark reported that regeneration of new phloem within 10 to 15 days resulted in translocation so nearly normal that no difference could be measured between control and treated shoots in analysis for sugar, starch, nitrogen, and ash. Murneek showed that apple fruit set was increased an equal amount whether girdling affected carbohydrate and nitrogen levels or not. Thus, carbohydrate and nitrogen levels appear to be of little importance in single, knife-cut girdling . Sax reports that phloem blocks induced by killing a small section of the stem with heat prevented the downward movement of vitamins, organic carbohydrates, and grow-regulators . Bonner , in girdling stems of tomato plants, found that in a few days there was an accumulation of thiamin, pyridoxine, pantothenic acid, riboflavin, sucrose, and nitrogen above the girdle. Lewis and McCarty state that it appears possible that the severance of phloem tissues causes a temporary increase or decrease in a growth regulator influencing fruit set. Much of the effects of girdling have thus been done with a single cut. However, other studies of girdling physiology have involved more severe cuts , including the removal of a band of bark from the tree trunk. This is what happens with a ring graft. With a ring graft the strip of bark which is removed from around the tree trunk is immediately replaced with a comparable sized strip of donor bark. The donor strip in this instance is replaced in the proper polar orientation. The ring graft can consist of a bark strip from sweet orange, sour orange, Rough lemon, sweet lime, trifoliate orange, Morton citrange, whatever the researchers’ choice. In unpublished data by the author, plastic electrical tape, or duct tape, was used to hold the donor bark strip in place and prevent desiccation. Since the procedure of a ring graft is somewhat more severe than a simple girdle, the healing process may be a little longer, perhaps weeks, or even months. When the ring of bark is removed from the parent tree, the bark separates at the cambial tissue. Enough cambial initials remain to form new xylem elements to the inside and new phloem tissues to the outside. The strip of donor bark inserted into the removed bark area also has cambial initials on its inner surface. Again,hydroponic vertical farming systems xylem elements are found to the inside, phloem elements to the outside . Roberts was one of the pioneers to explore the use of ring grafts in apples. The author was a graduate student of Roberts in the early 1940’s and recalls in several discussions that Roberts was quick to point out that the effects did not last since the donor bark strip soon became nonfunctional and sloughed off as with a cork cambium. Sax extensively investigated the aspect of bark inversion of the donor bark strip so that the polarity of the conducting cells is at least temporarily reversed and transport of nutrients, metabolites, etc. reduced. Sax considered inverted bark strips as phloem blocks. Reversed polarity is only a temporary problem in bark regeneration . Sax , referring to the work of Roberts , states that the inverted rings checked the growth of the trees, but without the deleterious effect resulting from girdling. He further emphasizes that apparently this work was not continued by Roberts. The author had the pleasure of a long discussion with Sax in 1957, and Sax admitted that the effects of bark inversion were temporary and had very little overall effect on tree growth. In 1956 , the author had conducted some bark inversion experiments with a grower cooperator. The trees were young lemon trees of uncertain destiny since the cooperator indicated the orchard would be removed in one to three years. Replicated treatments were made and the size of the tree trunks above the girdled area measured. The treatments consisted of different widths of inverted bark from 1/4 inch to 2 inches . In some treatments, height of the ring insertion varied. In other treatments, a double ring of bark was inverted, approximately 10-15 cm apart.

Another treatment consisted of an incomplete inverted ring with a 2.5 cm wide strip of normal bark at 180°. See Figures 11 – 21 [Image for Figure 21 could not be located]. Observations were limited because the orchard was pulled before final measurements could be made. In no instance was there any observable difference in tree growth with any treatment as compared to the checks. The width of the inverted ring did not seem to make any difference, the 5 mm was as effective as the 5 cm. Perhaps the use of two inverted rings had more effect than a single inverted ring. The most notable effect was with the incomplete rings where small islands of normal bark were left at 180°. The effectiveness of the blocks was clearly demonstrated since the nutrients and metabolites were translocated to the two normal bark strips resulting in accelerated growth increase in that area. The use of ring grafts using other varieties or species of citrus genetically different from the parent tree produces no permanent effect on the parent tree. Imposing the additional effect of bark inversion to alter polarity is also a temporary effect. Since nearly all commercial citrus trees are budded, it is not surprising that, like other budded or grafted fruit tree combinations, they may be subject to incompatibilities. The terms compatible and congenial refer to healthy long-lived combinations, and incompatible and uncongenial refer to combinations which are not healthy and long-lived. Sometimes the generalization is made that compatibility is a function of the closeness of genetic relationship between the stock and the scion, but there are certainly many exceptions to this hypothesis. If the generally accepted systems of citrus classification are used, there is a wide diversity in the germplasm available within the conservative classification of Swingle , who lists 16 species, and that of Tanaka , who lists 159 species. However, within the Family Rutaceae, subfamily Aurantioideae, there are a total of 33 genera available with considerable interspecific and intraspecific crosses and bigeneric and even trigeneric hybrids available. It is possible to graft any Citrus with Citrus, and apparently many genera with many other genera, but the degree of success may vary greatly. While the author is unaware of Citrus being successfully grafted on any genus outside the subfamily Aurantioideae, he has kept orange buds alive for one year when grafted on Casimiroa edulis, but the buds did not push. In a 1977 conversation with Professor Giovanni Fatta Del Bosco of Palermo, he indicated that mandarin scions will grow, but poorly, on this cultivar.

Seedlings of C. taiwanica, Yuma citrange and some of the Ichang hybrids, for example, also show a low rate of polyembryony and severe cullage is needed in the nursery. Cultivars with a low nucellar rate should probably not be used as root stocks. However, it is possible that if the nucellar seedlings are of considerable root stock interest, clonal propagation by in vitro methods could be considered in the future. In varieties that produce a fairly high percentage of nucellar embryos, most of the seedlings can be recognized as representing the largest proportion of the progeny. They will have the same foliage and branching characters and will in general be of about the same size. They present a uniform and, commonly, easily recognizable type. Mixed with these will be a varying number of seedlings that show different characters, such as different branching and larger or smaller leaves, and that are usually less vigorous and of smaller size. However, in some instances occasional seedlings are more vigorous than the norm. Variations in trifoliate hybrids may be more difficult to detect because the trifoliate leaf character is dominant and all the hybrids exhibit the trifoliate leaf character. Here, in addition, one must look for size of leaves, leaf color, the tendency towards deciduousness, and other factors as well. These differences from the norm, in a large part, are the so-called “variant” seedlings which apparently are mainly produced from gametic embryos. It is these small, off-type seedlings or extremely vigorous seedlings which are variants and should be discarded upon removal from the seedbed,vertical garden indoor or later in the nursery row, in order to obtain uniform seedlings for use as root stocks. In spite of all precautions, some zygotic seedlings do escape culling and are budded and ultimately end up as orchard trees.

Some of these, but not all, may be culled out as budlings in the nursery row because of lack of uniformity as compared to the rest of the progeny. It is unfortunate that most of the people involved in the labor or nursery practices are not trained or qualified to recognize variants. They probably do a better job on the elimination of “goose necks” and “bench roots,” which in reality may not be as important. Seedling progeny of some of the monoembryonic cultivars such as Algerian tangerine and a number of shaddock cultivars, although all genetically different, have been remarkably uniform in the seedbed and nursery. When budded to standard scion varieties and planted in root stock experiments at the Citrus Experiment Station, Riverside, most of the trees at 33 years of age were as uniform in size, appearance, and yield as those budded on nucellar seedlings of other varieties. Certain inherent characteristics persist in the progeny, however. Some seedlings of Algerian tangerine, shaddock, Poorman’s orange and others continue to show a variable percentage of the seedlings with a bark disorder. In testing numerous shaddocks as root stocks for tristeza resistance, the fact that every stock was genetically different did not influence the reaction to tristeza. All were susceptible. Many of the citrus relatives which are monoembryonic also display a high degree of uniformity when grown from seeds. Thus Clausena, Murraya, Triphasia, Citropsis, and other genera will, although possessing one zygotic embryo, produce progeny which appear phenotypically identical to the mother plant. In many of the world’s citrus areas, the importance of strain selection within scion varieties is well recognized and put into practice by the growers and nurserymen. This is perhaps more true in Japan than any other citrus area. Japan’s large Satsuma industry is divided into three fruit categories according to their maturity. The Wase group are early, the Nakates are mid-season, and the Futsu, or common group, are late. Within the Wase group are Okitsu, Miko, Miyagawa and dozens of others.

The other two groups may have a few less strains. Little differences in fruit yield, time of fruit maturity, hardiness, or local preference, etc., are of considerable importance to the grower and the industry without changing the variety. Spain has many strains of Clementines also selected for size, color, seediness, time of maturity, etc. Italy has made similar selections within the Willow Leaf mandarin. Strain selection within a variety has also been extensively practiced in California. Within the Washington navel variety are the Parent, Frost, Newhall, Tulegold, Bonanza, Thomson Imperial , Atwood, Fischer, Dream, Lane Late, etc. Even a wider range of strains may be found within the Eureka and Lisbon lemon varieties. These strain differences are for the growers’ preferential choice; the navels are all sold collectively as navels, and all the lemon strains are sold as lemons. Strain selection, or even varietal selection, while recognized within root stocks, is still generally not practiced, although there could be many advantages in doing so. Thus, for root stock purposes, the citrus industry has just nonchalantly considered a sweet orange a sweet orange, a sour orange a sour orange, or a Rough lemon a Rough lemon. On the other hand, root stocks like Troyer citrange, Sampson tangelo, Cleopatra mandarin, or Rangpur lime, are more specific; they imply one specific cultivar with no recognized variations, or strains, at least at the present time. A grower choosing one of these cultivars for root stocks doesn’t indicate to the nurseryman he wants citrange, tangelo, or mandarin as stock, because it is now a well known fact that there are other citranges, other tangelos, and other mandarins and, unless he specifies, he may get something else. Root stock trials at the Citrus Experiment Station, Riverside, indicate that there are performance differences between different strains of sweet oranges, sour orange, grapefruit, etc., that justify a clonal selection within that species to take advantage of greater yields, variations in tree size, better gummosis resistance, or better nematode resistance , and see root stock yield later in this manuscript. In these trials, and across several scion varieties, the performance of the Rubidoux sour and Brazilian sour was definitely superior to that on African sour and to a lesser extent on Paraguay sour.

Of course, this was before the incidence of Tristeza.The CRC #343 grapefruit provided better performance results than Duncan, McCarty, Camulos, and several others. The Koethen sweet orange in the Riverside trials appeared preferable to other sweet oranges. In Ventura County, the Olivelands sweet has frequently been preferred. The established performance of root stock cultivars warrants their perpetuation as root stock seed sources and their choice over untested and unproven sources which a nurseryman thinks may be just as good. In California, many citrus nurserymen have, or have had, their own individual seed source trees. Many had been chosen because of past good orchard performance in a specific area, or statewide. Some were the nurseryman’s selection,vertical plant rack others were obtained from the Citrus Research Center at Riverside. In either case, the nurseryman could offer the same clonal selection of root stock year after year with some confidence in its continued good performance. The immediate and rapid acceptance and success of Troyer citrange as a root stock in California almost had disastrous effects. With the increased plantings of young orchards, Troyer seed was in such demand that many nurserymen chose to grow seedlings for future seed sources. Prices were prohibitive for that time , as seed sold in most instances at a rate of ten cents each, or fifty cents per fruit, and three hundred dollars a standard field box . The author observed some of the fruit seedling sources and was able to identify a number of off type trees. The fact that some of the trees were off type didn’t seem to make any difference to the seed supplier. Fruit from the off type trees was harvested along with the fruit from the normal trees, either unknowingly or willingly , and sold as Troyers. While it is one thing to be able to identify any off type trees which can and should be discarded, another situation arises with off types in which there are no discernable visual differences from the mother tree. These may not be hybrid differences, but rather somatic variations which may be just as critical. The author will cite several examples, and keep in mind that this happened before the use of isozymes and other techniques were available for identification purposes. In the 1948 tristeza plantings at Baldwin Park, California , two sources of Bessie sweet orange were used as root stocks and half of the trees per root stock inoculated with tristeza. One of the sources of Bessie was an old source from the Citrus Variety Collection, Riverside. It served as the mother seed source for Bessie seedlings grown in 1924 for 1927 root stock plantings at Riverside . From out of these many seedlings, Dr. Webber had selected seedling #47 as being the largest and healthiest seedling of them all. This should have been the tip off. Seedling #47 was assigned CRC #1693 and also was placed in the Citrus Variety Collection, Riverside. In the Baldwin Park plantings, trees on the Bessie seedling CRC #1693 showed a reaction to tristeza and the trees on the Bessie #245 did not. Obviously the two seed sources were different. Many times my colleagues and I examined the two accessions and could not on leaf and fruit characteristics establish any differences. However, the largest and most vigorous seedling out of a progeny should never have been selected as true to type, and was probably either a gametic or somatic variant. Webber in 1924 also grew a fairly large number of sour orange seedlings for root stock purposes. Out of a population of 389 nursery seedlings, he selected 43 he considered as variants.

He propagated these variants and brought them into fruiting, and also budded the variants to a selected source of Washington navel. The author only wants to make reference to one of these variants. Just prior to my arrival in Riverside in 1946, one of the variants out of the 43 was selected for inclusion in the 1948 citrus root stock-tristeza trials at Baldwin Park . It was merely listed as a “sour orange variant.” The author was always interested in the correct identification of the root stock selections used in his experiments. Examination of this sour orange variant revealed leaf and fruit characters which appeared identical to Rough lemon. Showing foliage and fruit to my colleagues, including Dr. L. D. Batchelor, Dr. H. B. Frost, Dr. E. R. Parker and others, we could not differentiate between the variant and verified sources of Rough lemon. We removed the variant from the sour orange category and placed it in the lemon category, where we thought it more appropriately belonged. Upon being inoculated with tristeza, it proved to be equally, if not more, susceptible than those trees on sour orange. The standard Rough lemons showed no effect of the inoculation. Obviously, the variant was indeed a sour orange variant. How often does this happen? A number of the accessions in the Citrus Variety Collection date back to Dr. Webber’s “largest seedling” in the seedling nursery population. Many other accessions are more recent seedlings. Other seedling accessions may be more true to type than the Bessie, but do we know? Most of the accessions in the variety collection at Lindcove Field Station are seedlings, some of the accessions in the foundation block also. This is adequate cause for concern and alarm. Are they identical to the parent sources? A few years ago, Dr. John Carpenter of the USDA Date and Citrus Station at Indio, California, shocked a group of citrus nurserymen he was speaking to at Riverside, when he said many of you do not have Swingle citrumelo CPB 4475; what you have is a seedling source of Swingle citrumelo, and they are not the same. In recent years there has been considerable interest in C-32 and C-35 citranges . As information was developing and some credence given to the future use of these two root-stocks, some nurserymen obtained seed from Riverside and planted seedlings for future seed sources. The all important question remains, are these seedling sources identical to the original sources at Riverside, and to those tested at South Coast Field Station for tristeza resistance ?

Hanging or lifting irrigation lines where possible, and using covers over the spaghetti tubes, are effective methods to reduce rabbit damage to irrigation. In addition, nurseries may consider encouraging irrigation tubing manufacturers to develop spaghetti tubing more resistant to rabbit gnawing. Nurseries may want to consider an ongoing program to reduce rabbit harborages by using habitat modification where possible. It may be appropriate to target areas that unintentionally provide habitat to reduce suitability. Some measures to reduce influx and suitability may include fencing between natural areas/groves and the nursery; decreasing cover for the rabbits in runways; thinning border vegetation that provides cover, such as the hedges, to reduce hiding places; regularly moving debris piles, recycled wood piles, and storage pallets to reduce breeding and cover within the nursery; and rotating pallets of stock. In the circumstances when trapping is desirable to remove rabbits from an area, the use of traps in runways created by drift fence will be the most effective method. Two species of tree frogs native to the Caribbean have recently become established in the Hawaiian Islands . Since their introduction via the import horticultural trade, the frogs have rapidly expanded their range on the islands of Hawaii, Maui, Oahu, and Kauai. There are two modes of spread for tree frogs. The first is the accidental transport via horticultural products or material from infected nurseries or gardens to uninfected areas. The second mode is the intentional introduction of frogs by citizens into sites that aren’t infested. Theoretically both activities are illegal under Hawaii state law , though enforcement is difficult.

There is a concern on the part of federal, state,vertical farming technology and private agencies/entities managing natural and agricultural resources in Hawaii that introduced Eleutherodactylus frogs pose a serious threat to these resources. E. coqui can reach densities of greater than 24,000/ha and is capable of consuming approximately 114,000 arthropod prey items per hectare in a single night in its native range in Puerto Rico . It is believed that these frogs, once established in native habitats, could prey on endemic arthropods as well as compete indirectly and directly with native birds for limited food resources . Tree frogs may be a vector for plant nematode eggs, and the recent discoveries of frogs in certified nurseries make the frogs a potential quarantine issue that could greatly impact the exportation of disease and pest-free nursery products from the state. Surveys indicate that frog populations have become firmly established in nurseries, parks, residential gardens, resort areas, and lowland forest habitats on the islands of Hawaii and Maui . The number of reported locations has significantly increased on these islands in the last 3 – 4 years . Frog populations have been documented on the islands of Oahu and Kauai , and there is grave concern that these populations will continue to spread. In one horticultural site on the island of Hawaii, one species of tree frog has been documented to obtain densities comparable to the native range . Localized loud vocalization of male frogs throughout the nighttime hours has also been a source of numerous angry complaints from sleepless residents and tourists alike. Restricting the transfer of infected plant materials via the horticultural trade or by the casual public has the potential of stemming further spread of frogs to uninfected areas. However, enforcement of laws dealing with the transportation of alien species within the State of Hawaii has been limited at best. Currently, an enforceable legal mechanism that specifically restricts the movement of plant hosts harboring E. coqui within the state has been proposed by Hawaii Department of Agriculture staff but has not been enacted.

Though the status of an enforceable quarantine on the movement of frogs is currently in question, there is an immediate need to: 1) reduce or eradicate localized frog populations that serve as reservoirs for new infestations and 2) treat infested plant material to insure this situation does not get any worse. Since 1998, research has been conducted with the goal of developing control techniques for these frogs. Current trapping techniques proved in field trials to be inefficient . Cultural practices or hand capture may be effective on a small scale; however, chemicals appear to be the only broad-range and cost-effective immediate method of controlling frog populations. A laboratory study was conducted to screen 35 selected 1) pesticides registered for invertebrate control in ornamental nurseries and floriculture in Hawaii, 2) human pharmaceutical and food products, and 3) surfactants as potential chemicals for E. coqui and E. planirostris frog control . During initial screening, one of two commercially available pesticides containing resmethrin , a synthetic pyrethroid, was found to cause mortality to slightly greater than 50% of tree frogs tested at registered or recommended dosage rates . No surfactants tested were found to cause frog mortality rates greater than 50%. Of the human pharmaceuticals and food products tested, food grade caffeine , applied as a topical spray, proved effective against both E. coqui and E. planirostris. The lowest concentration solution of caffeine and water that resulted in 100% tree frog mortality was a 12.5 mg/ml solution applied to tree frogs topically with a spray bottle. Following the completion of laboratory screening of potential chemicals for Eleutherodactylus control, field trials were conducted on the directed spray application of three different caffeine solutions for controlling introduced Eleutherodactylus frogs in floriculture and nursery crops in Hawaii . The directed spray application of 0.5%, 1.0% and 2.0% caffeine solutions reduced Eleutherodactylus coqui abundance in test situations on or bordering infested ornamental plant nurseries in East Hawaii. Treatment of plots with a single spray application of a 2.0% concentration caffeine solution caused a 100% decline in the relative abundance of Eleutherodactylus frogs and in the relative abundance of frogs adjusted for nightly variation in frog activity before and after treatment on control and treatment plots.

The Hawaii Department of Agriculture’s Pesticide Branch prepared and submitted to the United States Environmental Protection Agency an application for an Emergency Registration for the spray application of caffeine for localized frog control in the State of Hawaii. The U.S. EPA granted the requested registration for a one-year period beginning on 27 September 2001 with the stipulation that data were collected on potential non-target impacts and monitoring of soil and ground water contamination concerns. There has been limited use of caffeine for frog control since the granting of the EPA registration, and the Hawaii Department of Agriculture’s Pesticides Branch has had to be revise and simplify data collection requirements considered cumbersome to potential users. Reporting requirements,vertical tower planter relatively high cost in comparison to other commercially available compounds for the control of pest insects and weeds, and limited data on non-target effects have been concerns raised by potential users of caffeine for frog control. Efforts have been made by other researchers and managers to identify additional compounds for frog control. Anecdotal laboratory observations and field applications suggest that the spray application of a concentrated hydrated lime and water solution may be an additional tool for frog control. As hydrated lime and water solutions are being sprayed on vegetation versus being applied to the ground as a soil supplement, this specific use pattern may need to be registered with the U.S. Environmental Protection Agency. Standardized data collected with some level of scientific rigor showing efficacy is needed to support the legal application of this solution for frog control. Other research is being conducted to determine if natural or synthetic pyrethroid compounds could be used for frog control . In late 2000, several species of potted plants were successfully treated for frogs in test situations using short term drenching with hot water . Since the first documentation of the presence of Eleutherodactylus frogs in Hawaii, populations have spread quickly and local abundance of frogs has grown dramatically, particularly in sites with higher levels of rainfall . Though this issue has garnered significant attention in the local and national media, efforts to control the spread of the frog have been limited. Frogs have, and will continue to, affect the quality of life of citizens who live in infested sites. Citizen frustration with a lack of progress dealing with this situation in infested residential and commercial sites has been building and will continue to build. There is a realistic concern that Eleutherodactylus frogs will be accidentally transported from Hawaii to mainland states within the United States and to other countries in infested cargo. Eleutherodactylus frogs are frequently found in Hawaii at retail nurseries associated with large department and hardware store chains. Eleutherodactylus frogs have been reported in California and Connecticut and these individual frog captures have been attributed to shipments of infested Hawaiian plant products . Though Eleutherodactylus frogs may not become established in many mainland sites within the United States, Eleutherodactylus infesting shipments within Hawaii or infesting shipments going to other Pacific Islands, tropical, and sub-tropical destinations internationally is a realistic quarantine concern. There is a clear need to establish if Eleutherodactylus frogs within certified nurseries are a potential vector for plant nematode eggs , as particular species of plant nematodes are a significant quarantine issue for plant products being shipped from Hawaii to the State of California. It is worth asking the question “why hasn’t there been a significant coordinated effort mounted to deal with introduced Eleutherodactylus frogs in Hawaii?” A primary reason is the lack of techniques to control the spread of frogs; hot water drenching was not tested as a quarantine technique until late 2000, and U.S. EPA emergency registration of the spray application caffeine and water solution for local control was not obtained until late 2001. It would be very hard to enact a quarantine on a pest that impacts a major industry without the ability to control the pest in a simple, cost effective manner. Lack of funding, unclear legal jurisdiction, and bureaucratic inertia are other reasons why the spread of the frogs has continued until it is unrealistic, given current progress dealing with this issue, to believe that frogs will be eradicated from the island of Hawaii . There have been significant efforts by individual staff members of various state, county, federal and non-governmental entities and private citizens to try to stem the spread of invasive frogs and eliminate local populations. USDA APHIS Wildlife Services staff and Hawaii Department of Agriculture staff have written a plan to control and/or eradicate frogs in the State of Hawaii . Funding for this plan is the most realistic means to deal with this situation. It is clear that decisive action to eradicate an introduced pest when it has a limited distribution is far less expensive than eradicating or controlling the pest when it is broadly established. Unfortunately, the window of opportunity for effective action against the Eleutherodactylus frogs in Hawaii is becoming shorter while the problems associated with these pests will become greater as more sites become infested and local frog abundance increases in previously infested sites. Exposure of susceptible plant tissues to non-freezing temperatures below 10–12 ◦C induces a physiological disorder called Chilling Injury . There appears to be two phases in the development of CI. The first phase is initiated in the cold and could involve a change in membrane fluidity or enzyme activity .These secondary symptoms are predicated by primordial events initiated in the cold, and include a host of metabolic and physiological changes that include increased membrane permeability , increased respiration and ethylene production , uneven ripening, disease susceptibility, water soaking and surface pitting . A technique that could detect the earliest physiological changes associated with CI would foster a better understanding of the initial events leading to this disorder, and point to more effective ameliorative action. Magnetic resonance imaging is a nondestructive imaging technique, which is increasingly used to visualize and quantify fruit physiological response to endogenous or exogenous stimuli . MRI uses the magnetic properties of nuclei and their interactions with radio frequency and applied magnetic fields to produce an image . Variations in the chemical composition and integrity of cellular structures can change the movement of water within and among tissues. These changes can be detected as modifications in the relaxation times of the protons in water, which in turn alters the signals used to construct MR images .