In a second replicated long-term field trial with ‘Valencia’ scion severely affected by HLB, highest yields over four harvests were with ‘US-942’ and ‘US-802’ root stocks. In both trials, some other root stocks that are commonly used in commercial production performed poorly, probably indicating less tolerance of those root stocks to HLB. Similarities and differences between the two trials will be discussed, as well as the value of different metrics for assessment of relative field tolerance of root stocks to infection by Las. The implications of these observations for the choice of root stocks to be used in commercial plantings will also be discussed. Interactions between plants and their associated microbial communities are complex, dynamic, and of varying impact. These interactions include plant-microbe and microbe-microbe relationships for each member of the community and can vary from positive to negative on each species. The structure of these communities can alter plant defense against other microbes and herbivores, plant productivity, and uptake of nutrients. Given these impacts, it is of interest to characterize the variation in microbial communities associated with crops facing major disease. The most devastating disease of Citrus is Huanglongbing caused by infection with at least one of Candidatus Liberibacter asiaticus, C. L. americanus, or C. L. africanus. Current methods of disease control involve disease vector suppression through conventional and bio-control methods, detection of infected trees, and inoculum removal.
Despite the success in slowing disease spread,growing strawberrries hydroponically these methods haven’t stopped the spread of disease. Methods to inhibit pathogen invasion may involve manipulations of microbial communities. In preparation for manipulative experiments, the microbial community of Citrus was characterized using 16S amplicon sequencing. Samples were collected from multiple locations within Florida, Texas, and California and consisted of trees cultivated using conventional and organic methods. Leaf midribs, fibrous roots, and soil were collected from trees infected with and trees with no detectable C. L. asiaticus. Results are discussed in terms of their impact on future microbial community manipulation experiments as well as their impact on disease detection. Liberibacter infection and tissue specificity. Several of these transcripts were selected as bait to detect protein-protein interactions using the yeast-2 hybrid system. Results indicate that transcripts involved in the immune response, endocytosis, and cytoskeleton assembly are important to Ca. Liberibacter infection of the psyllid host and/or in psyllid-mediated Ca. Liberibacter transmission. Ca. Liberibacter asiaticus is the obligate, fastidious bacterial pathogen that causes citrus greening disease, or Huanglongbing. CLas is transmitted in a circulative, propagative manner by Asian citrus psyllid Diaphorina citri Kuwayama adults. Identification and functional characterization of effectors involved in invasion of the psyllid gut and its presumed entry into the salivary glands was investigated using transcriptomic, proteomic, yeast-2 hybrid and coimmunoprecipitation analyses. In silico annotation and differential expression analysis of contigs from ACP nymphs and adults, and adult midgut and salivary gland tissues identified transcripts and proteins with altered expression in response to CLas infection. Several differentially expressed transcripts were selected and used as bait for Y2H detection of protein-protein interactions.
Those positive by Y2H were subjected to verification by bait to prey co-transformation and CoIP. Proteins positive in one or both assays were tested in ‘knock down’ experiments using dsRNA to induced RNA-interference and quantified by qPCR. Candidates of the greatest interest have been those with a predicted role in virulence and invasion, that if disrupted by RNAi could abate CLas accumulation in and exit from the gut, circulation in hemolymph and systemic infection, and acquisition e.g. in salivary glands. Collectively, the results suggest a model for invasion in which CLas- and prophage-encoded effectors transform the endocytic host pathway into a ‘pathogen-mediated phagocytic scenario’ utilizing membrane ruffling in CLas interactions with the gut, leading to bacterial exit into the hemocoel and systemic invasion of other psyllid host tissues and organs. Ca. Liberibacter asiaticus and solanacterum are the causal agents of Huanglongbing and Zebra chip disease of citrus and solanaceous crops, respectively. Bacterial effectors that interact with psyllid vector proteins during invasion of and exit from the gut, and that facilitate entry into the salivary glands were identified using functional genomics, proteomics, yeast-2 hybrid , co-immuno precipitation , and electron microscopic analyses. In silico annotation and differential expression studies of psyllid nymphs and adults, and midgut and salivary glands identified transcripts and proteins with altered expression in response to Ca. Liberibacter infection. Differentially expressed transcripts were used as bait in Y2H studies to corroborate protein-protein interactions. Those positive by Y2H were subjected to verification by bait to prey co-transformation and Co-IP. Electron micrographs of the PoP midgut revealed the presence of CLso in the ventricular lumen, apical and basal epithelial cytosol, and in the filter chamber periventricular space.
CLso were also prevalent in salivary gland pericellular spaces and head epidermal cell cytosol. Collectively, the results suggest a model for invasion in which Ca. Liberibacter- and prophage-encoded effectors manipulate the endocytic host pathway into a ‘pathogen-mediated phagocytosis scenario’ leading to bacterial exit from the gut into the hemocoel, and systemic invasion of other psyllid host tissues and organs. Beyond the devastating effects of HLB on tree health and crop size are the secondary impacts of preharvest fruit drop and the quality degrading flavor profile of symptomatic fruit. A precise number for the increasing percentage of the smaller, symptomatic fruit being sent to processors for juice extraction is not readily available. But inspection of data provided by NASS does indicate the number of fruits per box has been trending higher over the last five seasons. As previously noted increasing disease severity will increase the percentage of symptomatic fruit being used for juice extraction. In a 2015 survey of growers, production managers and other associated occupations 81% of the responders reported that 80% – 100% of their acreage was infected and the average percent of infected trees was 90%. As more and more fruit become symptomatic their detrimental effect on juice quality will become more and more pronounced. Beyond a blending ratio of 3:1 off-flavors in the product become discernible. The incidence of pre-harvest fruit drop also has been increasing with a recently reported 10% – 20% rise. Enabling a technology to utilize these two groups of HLB damaged fruit and recover lost value would be beneficial to the citrus industry during this transition period before resistant trees are available, especially if that technology also could provide increased value for all juice extracted biomass. Here we report on a pilot-scale continuous process to release and recover multiple value added co-products from culled fruit, preharvest dropped fruit and juice extracted biomass. Using steam-explosion followed by a simple water wash we have been able to recover pectic hydrocolloids, bioactive phenolics and fermentable sugars.Pectic hydrocolloids are functional polysaccharides that can be used to modify the rheology of water and as a hydration control or ion capture agent. Citrus phenolics contain a number of compounds that possess bioactivity against chronic human illnesses. Sugars can serve as a feed stock for a variety of synthetic options as well as an energy source in varied fermentation schemes. Limonene has a variety of established commercial applications. Summing these potential co-products could promote the dropped or flavor degrading fruits as an alternative value added resource and recover increased value from juice extracted biomass vs. a traditional feed mill operation.Huanglongbing constitutes the most destructive disease of citrus worldwide,rolling bench yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, ‘Candidatus Liberibacter asiaticus’. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified.
The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB. Tamarixia radiateestablished and spread throughout Florida following first release in 1999 for control of the Asian citrus psyllid Diaphorina citri . Nevertheless, persistence of the pest and continued losses from citrus greening disease have created the need for more effective biological control, including mass rearing and augmentative release of T. radiata. However, information is lacking on ways to manipulate host density to optimize rearing efficiency. Three release rates of D. citri per shoot, and four host: T. radiate ratios were evaluated. Results indicated that 20 psyllid adults per flush provided optimal host density without excessive stickiness from honeydew. Sixty T. radiate females per cage of approximately 4,800 nymphs provided most economical use of wasps and hosts. However, only 60% of the hosts were utilized for progeny production under these conditions, possibly due to competition between females mediated through deterrent host marking. In order to determine the existence of a host mark on parasitized hosts, a three-day-old T. radiate females was released in a petri dish with six parasitized hosts and six clean hosts randomly distributed on an orange jasmine shoot. The number of hosts probed and parasitized in each host category was noted. To evaluate volatility of the host mark, 10 parasitized 4th instar D. citri nymphs and 10 clean nymphs were randomly assigned to one of the arms of the T- maize olfactometer. Ten T. radiate females were released at the releasing chamber to make a choice of the two odor sources. Results showed that T. radiate females probed and parasitized significantly more on the clean hosts versus parasitized hosts but showed no a preference for either of the odor sources. Thus, T. radiate females were able to discriminate between clean hosts and host parasitized by herself by perceiving a non-volatile host mark. Future research will focus on determining whether the mark also has a deterrent effect on nearby hosts. The ultimate goal is to improve mass rearing efficiency and biocontrol effectiveness in the field. Candidatus Liberibacter asiaticus is the bacteria associated with the economically-important citrus disease, Huanglongbing . Understanding the pathogenicity of CLas and its effects on the citrus host is challenging, as CLas has yet to be cultured. To understand the impact of CLas infection on host leaf metabolism, Parent Washington Navel orange Osbeck greenhouse plants were graft-inoculated with CLas+ or CLas- budwood, and leaf samples were collected longitudinally until tree death. Samples were analyzed with metabolomics , transcriptomics , and proteomics to identify CLas-associated changes in the plant. Changes in metabolite content could be observed as soon as 6 weeks post-infection, before symptom development. Further changes in the metabolome, transcriptome, and proteome were observed throughout the study, with the most drastic differences noted at the last time point as the trees were nearing death. CLas affected both primary and secondary metabolism, including photosynthetic processes, cell wall synthesis, and stress responses. These data show that early changes in tree metabolism occur in asymptomatic plants with CLas infection, and shed light on how CLas impacts the citrus host throughout the disease. The concept of growing degree-day , which integrates the rate of development of an organism as a linear function of air temperature to which it is exposed, has been widely used in studies of crop and insect phenology for scheduling agricultural practices. In citrus, however, these studies have focused on the reproductive development and on the physicochemical characteristics of the fruits but not on vegetative shoot ontogeny. This is important because vegetative shoots are strictly linked to the biological performance of Diaphorina citri. Although the thermal requirements of the psyllid have been established in growth chambers, little is known about the thermal requirements for the psyllid in natural environments or about the cumulative heat units for the emission and development of new shoots, taking into consideration their ontogeny. To assess this, experiments were carried out in growth chambers using 2.5 year-old potted ‘Valencia’ orange grafted on ‘Swingle’ citrumelo. The plants were pruned 20–30 cm above the grafting line and evaluated for several flushing cycles. Daily assessments of shoot size, new leaf emergence and leaf area were carried out on a single shoot per plant.