Two clones remained negative after challenges and a third had a low percentage infection. The ultimate evaluation will be in the field where they are exposed to repeated natural infection by the insect vector. Trees of the three clones were planted in November 2007 amongst old trees with greening symptoms. This report is on their current status regarding greening infection as well as growth, production and fruit quality.The objective was to evaluate soil-applied SAR inducers or transgenic enhancement of SAR expression for effect on HLB disease progress in citrus trees challenged by graft-inoculation or psyllid-mediated infection. One yr-old Hamlin sweet orange trees were planted in May 2009 and treated as follows: 1) non-treated check , 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. A randomized block design was used with 5 repetitions of 10 tree plots. In 2010, the SAR inducer acibenzolar-S-methyl which does not control psyllids was substituted in treatments 3, 4 and 6. At 24 months after treatments were established in a field site, 105 trees were PCR+ in the trial. Higher numbers of PCR+ trees occurred in the UTC, the UTC with graft inoculation, and the IMID/THIA/ASM with graft-inoculation. A lower number of PCR+ trees occurred in the treatments with SAR inducers, foliar insecticides, and foliar insecticide plus SAR inducers,vertical farming racks but no effect of SAR treatment on rate of HLB disease progress was detected. Transgenics of Duncan grapefruit and Hamlin trees were constructed that expressed the NPR1 gene from Arabidopsis , a key positive regulator of SAR.

Over-expression of AtNPR1 in transgenic lines of these susceptible hosts reduced citrus canker lesions. Resistance to Xanthomonas citri subsp. citri infection was related to expression levels of AtNPR1. Two lines each of ‘Duncan’ grapefruit and Hamlin with the highest expression of AtNPR1 were screened in a greenhouse containing HLB-infected plants and psyllids. Rate of infection progress over 12 months was similar for transgenic lines and non-transgenic controls. Again, an effect of SAR on HLB disease progress was not-detected which confirmed the ineffectiveness of enhanced SAR for HLB disease control. Trioza erytreae is in itself a minor pest of citrus, but its significance is attributed to its ability to vector the citrus African Greening disease pathogen, Candidatus Liberibacter africanus. The population fluctuation of the citrus triozid is correlated to the flushing rhythm of the citrus host, but seasonal fluctuation in infection of populations carrying the Greening pathogen have been difficult to monitor in the past due to limited detection methodologies. This study explored this fluctuation in infectivity in an orchard entirely infected with African greening by using PCR to test individual triozids caught on sticky traps placed weekly in the orchard. Two season’s data are presented from a small sour orange orchard in the Nelspruit district in South Africa. The triozid population fluctuations correlated to previous findings of population peaks following the citrus flush cycles and responses to specific climatic influences. Fluctuations in the percentage infectivity of the T. erytreae populations were observed, with infectivity peaking at or just after the citrus flush seasons, but with peaks in infectivity differing to population peaks.Over the last decade the plant disease Huanglongbing has emerged as a primary threat to citrus production worldwide. HLB is associated with infection by one of a group of phloem-limited bacteria that are readily transmitted by the Asian citrus psyllid, Diaphorina citri.

However, the temporal progression of infection, time to symptom expression , and how they relate to transmissibility remains unresolved. We graft inoculated sweet orange trees with C. Liberibacter asiaticus, then at different times after inoculation, we inspected plants for HLB symptoms, measured bacterial infection levels in plants, and measured acquisition by psyllid adults that were confined on the trees. Plant infection levels increased rapidly over time, saturating at uniformly high levels near 200 days after inoculation – the same time at which all infected trees first showed disease symptoms. Pathogen acquisition by vectors was positively associated with plant infection level and time since inoculation, with acquisition occurring as early as the first introduction 60 days after inoculation. These results suggest that there is ample potential for psyllids to acquire the pathogen from trees during the asymptomatic phase of infection. If so, this could limit the effectiveness of tree rouging as a disease management tool and would likely explain the rapid spread observed for this disease in the field. The diseases Huanglongbing and citrus tristeza are both phloem limited and have significant economic impact on citrus production wherever they are found. Studies of host resistance have indicated that Poncirus trifoliata has tolerance/resistance to both diseases, suggesting there may be some common factors in the two kinds of resistance. We have conducted studies of host gene expression changes that occur in response to infection to gain further insight. Controlled inoculation by grafting infected budwood was used to infect potted greenhouse plants of Cleopatra mandarin , US-897 , and US-942 with CTV and with Candidatus Liberibacter asiaticus, the pathogen associated with HLB. Stem and leaf tissues was collected at 10, 20, and 30 weeks after inoculation. DNA-free RNA was subjected to real-time one-step SYBR Green RT-PCR analysis and relative gene expression was determined using the 2–ΔΔCt -method. Differences in gene expression between mockinoculated and Citrus tristeza virus -inoculated plants were generally not prominent. Differences in gene expression between mock-inoculated and Lasinoculated plants were most pronounced in susceptible Cleopatra plants and at the later stages of infection. Transcripts for a constitutive disease resistance protein were induced in response to Las in susceptible plants, but showed higher expression levels independent of infection in the tolerant genotypes.

A gene for a Myb-like transcriptional regulator family protein which is associated with resistance to some phytoplasma, responded strongly to Las, but also responded to CTV in tolerant plants. Transcript levels for other genes, such as a 2-oxoglutarate and Fe- dependant oxygenase and a plastidic glucose transporter , were considerably higher in US-897 and US-942 plants compared with Cleopatra plants independent of infection with either pathogen. It is hypothesized that these genes play a role in the resistance or tolerance of trifoliate orange and its hybrids to HLB and CTV.The Asian citrus psyllid is fairly well established in all counties in southern California and is spreading up the coast into Ventura and Santa Barbara Counties, but is not established in the major citrus production area of the Central Valley. The current status of Huanglongbing in California remains as only one backyard tree in Los Angeles County. This tree was identified as being infected with Candidatus Liberibacter asiaticus in March 2012 and removed. Despite continued surveys in that area,vertical rack system no additional trees have been found positive for HLBassociated pathogens.As a means of early diagnosis, adult and nymph ACP are collected and tested to determine whether HLB-associated bacteria are present in a localized area. The current approved diagnostic method for HLB in the United States is q-PCR; however, as demonstrated in Florida, this method may detect HLB-associated bacteria too late for the implementation of an effective eradication strategy. Hence, the Citrus Research Board has made the development of early HLB diagnostic methods their highest research priority. Methods being pursued involve the detection of molecules or compounds unique to Liberibacter infection within the host plant including volatile organic compounds , proteins secreted by the bacteria, small RNAs, and metabolomic compounds. These methods are very promising and are being tested against the approved q-PCR assay in time-course experiments conducted within a containment facility on the University of California, Davis campus. In addition, the Jet Propulsion Laboratories/NASA will monitor the reflectance properties of leaves for changes during the course of an infection. If this method is successful, JPL will further evaluate whether a large scale survey can be conducted by air or satellite. These new methods are also being field tested in Los Angeles County in the neighborhood where the CLas-infected tree was found in 2012 and in Texas in the vicinity of previous detections of HLB-positive trees. Candidatus Liberibacter is a genus of plant entophyte gram-negative bacteria in the Rhizobiaceae family vectored by psyllids. Most of the members of the genus are associated with damaging plant diseases and are subject to phytosanitary regulation. Ca. L. asiaticus, Ca. L. africanus and Ca. L. americanus are associated with Huanglongbing, one of the most destructive diseases of citrus. The disease is present in different countries of Asia, America and Africa but the three pathogens and their vectors Diaphorina citri and Trioza erytreae have a more complex distribution. The disease continues to spread rapidly and is a threat to the Mediterranean Basin countries, where the disease has not been reported. Early detection of the citrus Liberibacter in host plants and vector insects is essential to prevent its introduction and spread and for HLB control and management in countries where the disease is present. Diagnostic methods have to cope with the uneven distribution of the bacterium in plant tissues associated with irregular vector transmission, low bacterial titer during early stages of infection in host plants and vector insects and require differentiation of the individual species. By using the InCheck Platform , which combines multiplex PCR and microarray hybridisation on a single chip, we are developing a diagnostic assay to identify and discriminate among the species of Liberibacter in plant DNA extracts. The design of the probes on the microarray module for the identification and discrimination was based on the sequences of the following species: Ca. L. asiaticus, Ca. L. africanus, Ca. L. americanus, Ca. L. solanacearum, Ca. L. europeus, Ca. L. crescens and outgroup species within the Rhizobiaceae family.

The theory of natural spread of psorosis by insects in Argentina was stated by Rodríguez Pujol and Beñatena, researchers from the Concordia Experiment Station of the National Institute of Agricultural Technology , in 1965. In 1966, Rodríguez Pujol mentioned natural spread of psorosis in seedlings in the citrus region along the Uruguay River in Argentina. Timmer and Beñatena reported 0.1 – 1% of the root stock seedlings in the nursery were rogued because of psorosis. Timmer and Garnsey observed in Texas natural spread of Citrus ringspot virus in grapefruit at a rate less than 2%. Beñatena and Portillo showed psorosis transmission from diseased plants to healthy citrus seedlings by Toxoptera citricidus and T. aurantii winged individuals, by wingless adults of Toxoptera spp., by colonies of Toxoptera spp. and Aphis citricola, with a long incubation period and erratic symptom appearance. Since then, there have been advances in the study of the causal agent, diagnostic methods, and possible transgenic lines tolerant and /or resistant to CPsV but little has been mentioned about the natural spread of this virus. This work confirms the aerial spread by insects of CPsV in the field, both in a plot of certified citrus plants free of CPsV and in trials for testing aerial and/or soil spread of the virus in the Uruguay River citrus region in Argentina.Citrus psorosis virus is the causal agent of Psorosis, an important disease causing significant economic losses in Argentina and Uruguay. CPsV is the type member of the genus Ophiovirus, family Ophioviridae. Its genome has three singlestranded RNAs of negative polarity, encapsidated with a coat protein . As there is no natural resistance to Psorosis, transgenic Pineapple sweet orange plants were obtained expressing an intron-RNA hairpin from the cp gene . This RNA transcript induces the post-transcriptional gene silencing mechanism, degrading all RNA with cp gene sequences, thus preventing the progress of the infection. When ihpCP citrus trees were CPsV-challenged by graft-transmission, at least two SwO lines did not show any symptoms and CPsV was not detected by molecular and serological analysis. The hallmark of the PTGS is the presence of small RNAs , which were detected in the transgenic tissues indicating that resistance is PTGS-mediated. Our interest is to evaluate whether ihpCP is able to transfer this feature of resistance to susceptible grafted-tissues. For that, non-transgenic buds were grafted on the two resistant ihpCP lines used as rootstocks, which had previously been inoculated with CPsV. In another assay, a susceptible CP-line, expressing the complete CP-mRNA, was grafted on the same two ihpCP resistant rootstocks. Both, the non-transgenic and transgenic CP were susceptible. New flushes showed characteristic symptoms and TAS-ELISA and qRT-PCR analysis were positive.