Horticulture

Abstract Candidatus Liberibacter asiaticus (Las) is one of the causal agents of huanglongbing (HLB), the most devastating disease of citrus worldwide. Due to the intracellular lifestyle and significant genome reduction, culturing Las in vitro has proven to be extremely challenging. In this study, we optimized growth conditions and developed a semi-selective medium based on the results of nutritional and antibiotic screening assays. Using these optimized conditions, we were able to grow Las in the LG liquid medium with ca.100- to 1000-fold increase, which peaked after 4 to 6 weeks and were estimated to contain 106 to 107 cells/ml. The cultured Las bacteria remained in a dynamic state of growth for over 20 months and displayed limited growth in subcultures. The survival and growth of Las was confirmed by fluorescence in situ hybridization with Las-specific probes and expression of its metabolic genes. Growth of Las in the optimized medium relied on the presence of a helper bacterium, Stenotrophomonas maltophilia FLMAT-1 that is multi-drug resistant and dominant in the Las co-culture system. To recapitulate the disease, the co-cultured Las was inoculated back to citrus seedlings via psyllid feeding. Although the Las-positive rate of the fed psyllids and inoculated plants were relatively low, this is the first demonstration of partial fulfillment of Koch’s postulates with significant growth of Las in vitro and a successful inoculation of cultured Las back to psyllids and citrus plants that resulted in HLB symptoms. These results provide new insights into Las growth in vitro and a system for improvement towards axenic culture and anti-Las compound screening.

Huanglongbing (HLB) is a major disease of citrus associated with phloem-limited bacteria in the genus Candidatus Liberibacter that affects all known citrus species and relatives, with many commercial cultivars being greatly damaged. Testing cultivar tolerance to HLB in field conditions is difficult because of the erratic spread of the bacteria, scion and rootstock interactions, and influence of many biotic and abiotic factors on the tree response to the disease. This study aimed to validate the effect of CLas infection on different citrus species and hybrids thought to have different levels of tolerance to the disease using CLas graft inoculation under controlled greenhouse conditions. Young potted seedlings from 12 different citrus germplasm selections were graft-inoculated with CLas or mock-inoculated. Plants were monitored periodically during 18 months for canopy growth, HLB and nutritional leaf symptoms, and leaf CLas titers. The leaf nutrient content was measured at the end of the experiment. Roots were also assessed at 18 months after inoculation (mai) for CLas titers and biomass distribution. There were significant differences in most analyzed variables of healthy and infected plants. Some plants of all cultivars were successfully infected; however, overall, the CLas transmission rate was low and inconsistent. Ct values of roots were generally higher than those in leaves at 18 mai. HLB symptoms were not observed on seedlings until 1 year after inoculation; at 18 mai, infected trees of all cultivars were HLB symptomatic. Significant shoot and root biomass reductions (44%–75%) in infected ‘Cleopatra’, ‘Duncan’, ‘Olinda Valencia’, ‘Sunburst’, and ‘Valencia 1-14-19’, considered susceptible to HLB, were measured. These cultivars also showed more severe HLB symptoms than the presumed tolerant cultivars such as Microcitrus inodora, Rich 16-6 trifoliate orange, and US-897. This study provides new knowledge of the efficacy and value of greenhouse screening of citrus germplasm for response to HLB to support the development of new cultivars with improved HLB tolerance or resistance.

AbstractInfection with the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) is suspected to cause severe damage in carrot leading to high carrot weight loss. This study investigates three main aspects: (i) whether there is a reduction of carrot root weight under field conditions due to Lso infection; (ii) the correlation between Lso infection rate in carrot plants and occurrence of the psyllid Trioza apicalis as the known vector for Lso in carrot, and (iii) the comparison between symptoms described in literature and observed symptoms associated with Lso infection. Therefore, field surveys were conducted from 2018 to 2021 on organically managed carrot fields in Lower Saxony, Germany. Two Rebell orange sticky traps were placed per field replaced on a weekly basis. Captured T. apicalis were morphologically identified and counted. Carrot plant samples were collected from T. apicalis infested fields. Discolouration of foliage and further symptoms on carrots were investigated. For detection of Lso in carrot plants and psyllids, the samples were analysed using PCR. The infection rate of carrot plants varied between 2.5% and 80% per field with low abundance of T. apicalis. Of the asymptomatic plants, 39.4% were infected with Lso, while 80.9% of the symptomatic plants were infected with Lso. The weight of Lso-positive carrot samples was not significantly reduced compared to Lso-negative samples. No economically relevant losses of carrot yield were reported by farmers participating in this study. This indicates that there was no effect of Lso infection on carrot root weight during this study. Of the symptoms associated with a Lso infection in carrots, only leaf discolouration could be confirmed. Overall, the bacterium is present in organically grown carrot plants in Lower Saxony but no major carrot root weight losses were observed.

Abstract Huanglongbing (HLB) primarily caused by Candidatus Liberibacter asiaticus (CLas) has been threatening citrus production globally. Under HLB conditions, an excessive accumulation of the polysaccharide callose in citrus phloem occurs, leading to phloem blockage and starch accumulation in leaves. The callose production is controlled by callose synthases (CalS), which have multiple members within plants. However, the knowledge of callose production in the citrus upon infection with CLas is limited. In this study, we firstly identified 11 CalSs in the Citrus sinensis genome through bioinformatics and found the expression pattern of CsCalS11 exhibited a positive correlation with callose deposition in CLas-infected leaves (correlation coefficient of 0.77, P ≤ 0.05). Knockdown of CsCalS11 resulted in a reduction of callose deposition and starch accumulation in CLas-infected citrus. Interestingly, we observed significantly higher concentrations of abscisic acid (ABA) in HLB-infected citrus leaves compared to uninfected ones. Furthermore, the expressions of CsABI5, CsPYR, and CsSnRK2 in the ABA pathway substantially increased in citrus leaves upon CLas infection. Additionally, the expression of CsCalS11 was significantly upregulated in citrus leaves following the application of exogenous ABA. We confirmed that CsABI5, a pivotal component of the ABA signaling pathway, regulates CsCalS11 expression by binding to its promoter using yeast one-hybrid assay, dual luciferase assay, and transient expression in citrus leaves. In conclusion, our findings strongly suggest that the CsABI5-CsCalS11 module plays a crucial role in regulating callose deposition through the ABA signaling pathway during CLas infection. The results also revealed new function of the ABA signaling pathway in plants under biotic stress.

AbstractRecent outbreaks of ‘Candidatus Phytoplasma solani’ resulted in severe losses in potatoes, vegetable crops and grapevines in certain regions of Austria and constituted a major challenge for seed potato production. Therefore, the effects of various insecticides and insect deterrents on pathogen spread were studied both in laboratory and field experiments from 2018 to 2021. In laboratory transmission experiments, field captured Hyalesthes obsoletus were caged on differently treated Catharanthus roseus for five days. The insecticides lambda-cyhalothrin, deltamethrin, esfenvalerate, acetamiprid and chlorpyriphos showed the most rapid impact on insect survival and fully prevented phytoplasma transmission. The particle film forming products kaolin and diatomaceous earth had some effect. A transfer of the promising laboratory results to potato fields, however, was achieved to a limited extent only. Treatments with pyrethroids and acetamiprid every 8–10 days over the flight period of H. obsoletus roughly halved the number of symptomatic plants and tubers in case of moderately susceptible varieties and moderate infection pressure. In the event of susceptible varieties and high disease pressure, treatment effects were hardy discernible. In practical terms, the experiments indicate that insecticide applications alone are not sufficient to mitigate the disease. Spraying of diatomaceous earth and mineral oil did not affect disease incidence in the field.

Abstract The most damaging citrus diseases are Huanglongbing (HLB) and citrus canker, which are caused by Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. citri (Xcc), respectively. Endolysins from bacteriophages are a possible option for disease resistance in plant breeding. Here, we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas. LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc, according to inhibition zone analyses. The two genes, driven by a strong promoter from Cauliflower mosaic virus, 35S, were integrated into Carrizo citrange via Agrobacterium-mediated transformation. More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB, allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term. LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc, indicating that LasLYS2 had dual resistance to HLB and citrus canker. A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations, which might boost the citrus defense response, according to transcriptome analysis. We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2. Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2. Overall, the work shed light on the mechanisms of resistance of CaLas-derived endolysins, providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.

Stone fruits are a multibillion-dollar industry for the United States and Canada, one that has repeatedly suffered significant economic losses due to outbreaks of the X-disease phytoplasma (‘ Candidatus Phytoplasma pruni’) over the last century. Orchards and entire production areas have been abandoned, with corresponding losses to growers, fruit packers, and consumers. The most recent outbreak, in the U.S. Pacific Northwest, resulted in an estimated $65 million (USD) loss in revenue between 2015 and 2020 and is only increasing in incidence. Already present across much of the continental United States and Canada, the phytoplasma has a broad host range beyond stone fruit and is transmitted by at least eight leafhopper species, therefore stone fruit production in every state is at significant risk. This recovery plan was produced as part of the National Plant Disease Recovery System and is intended to provide a review of pathogen biology, assess the status of critical recovery components, and identify disease management research, extension, and education needs.