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‘Candidatus Liberibacter asiaticus’ is associated with the devastating citrus disease Huanglongbing (HLB). It is transmitted by grafting infected material to healthy plants and by the feeding of the Asian citrus psyllid (Diaphorina citri). Previously, we demonstrated that a metabolomics approach using proton-nuclear magnetic resonance spectroscopy discriminates healthy from diseased plants via grafting. This work assessed the capability of this technology in discriminating healthy and diseased plants when the bacterium is vectored by psyllids. One-year-old greenhouse-grown ‘Lisbon’ lemon trees were exposed to either carrier psyllids (exposed, n = 10), or psyllids that were free of ‘Candidatus Liberibacter asiaticus’ (control, n = 6). Leaf metabolites were tracked for 1 year and disease diagnosis was made using quantitative PCR. Overall, 31 water-soluble metabolites were quantified in leaves, including four sugars and 12 amino acids. Analysis via nonmetric multidimensional scaling and principal component analysis revealed significant differences between the leaf metabolome of control versus infected trees beginning at 8 weeks postexposure, including alterations in glucose and quinic acid concentrations. These findings provide a longitudinal overview of the metabolic effects of HLB during the early phases of disease, and confirm previous experimental work demonstrating that infection elicits changes in the leaf metabolome that enables discrimination between healthy and infected plants. Here we demonstrate that the mode of inoculation (i.e., graft versus psyllid) results in a similar pathology.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon (Citrus limon) plants were graft-inoculated with either CLas-infected or control (CLas-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with CLas infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between CLas-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of CLas-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Huanglongbing (HLB) is a severe, incurable citrus disease caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas). Although citrus leaves serve as the site of initial infection, CLas is known to migrate to and colonize the root system; however, little is known about the impact of CLas infection on root metabolism and resident microbial communities. Scions of ‘Lisbon’ lemon and ‘Washington Navel’ orange grafted onto ‘Carrizo’ rootstock were grafted with either CLas-infected citrus budwood or uninfected budwood. Roots were obtained from trees 46 weeks after grafting and analyzed via 1H nuclear magnetic resonance spectroscopy to identify water-soluble root metabolites and high-throughput sequencing of 16S rRNA and ITS gene amplicons to determine the relative abundance of bacterial and fungal taxa in the root rhizosphere and endosphere. In both citrus varieties, 27 metabolites were identified, of which several were significantly different between CLas(+) and control plants. CLas infection also appeared to alter the microbial community structure near and inside the roots of citrus plants. Nonmetric multidimensional scaling (NMDS) and a principal coordinate analysis (PCoA) revealed distinct metabolite and microbial profiles, demonstrating that CLas impacts the root metabolome and microbiome in a manner that is variety-specific.

The Asian citrus psyllid (ACP) is a phloem-feeding insect that can host and transmit the bacterium Candidatus Liberibacter asiaticus (CLas), which is the putative causative agent of the economically important citrus disease, Huanglongbing (HLB). ACP are widespread in Florida, and are spreading in California; they are the primary mode of CLas transmission in citrus groves. To understand the effects of ACP feeding, different numbers of ACP [0 ACP (control), 5 ACP (low), 15–20 ACP (medium), and 25–30 ACP (high)] were allowed to feed on Citrus macrophylla greenhouse plants. After 7 days of feeding, leaves were collected and analyzed using 1H NMR. Metabolite concentrations from leaves of trees with ACP feeding had higher variability than control trees. Many metabolites were higher in concentration in the low ACP feeding group relative to control; however, leaves from trees with high ACP feeding had lower concentrations of many metabolites relative to control, including many amino acids such as phenylalanine, arginine, isoleucine, valine, threonine, and leucine. These results suggest ACP density-dependent changes in primary metabolism that can be measured by 1H NMR. The implications in plant defense are discussed.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important pest in the worldwide citrus industry. It is the vector of “Candidatus Liberibacter asiaticus,” the bacterial pathogen of Huanglongbing, which is currently considered the most destructive disease of citrus worldwide. DcRV was previously identified based on metagenomics surveys for virus discovery. Here, we found that this novel and persistent insect reovirus took advantage of a virus-encoded nonstructural protein, P10, for efficient vertical transmission from parents to progeny. P10 assembled into a virion-packaging tubular structure and was associated with oocytes of female D. citri and sperm of males. Consistent with this, knockdown of P10 for either male or female D. citri insects inhibited DcRV transmission to offspring. This tubular strategy for viral spread and biparental transmission might serve as a target for controlling viral vertical transmission and population expansion., Diaphorina citri reovirus (DcRV) was previously identified based on metagenomics surveys for virus discovery. Here, we demonstrated that DcRV induces persistent infection in its psyllid host, Diaphorina citri. DcRV was efficiently vertically passed to offspring in a biparental manner. Transmission electron microscopic and immunological analyses showed that the DcRV-encoded nonstructural protein P10 assembled into a virion-packaging tubular structure which is associated with the spread of DcRV throughout the bodies of D. citri insects. P10 tubules containing virions were associated with oocytes of female and sperm of male D. citri insects, suggesting a role in the highly efficient biparental transmission of DcRV. Knocking down P10 by RNA interference for males reduced the percentage of DcRV-infected progeny and for females reduced the viral accumulation in progeny. These results, for the first time, show that a nonstructural protein of a novel insect reovirus provides a safe and pivotal channel for virus spread and biparental transmission to progeny. IMPORTANCE The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important pest in the worldwide citrus industry. It is the vector of “Candidatus Liberibacter asiaticus,” the bacterial pathogen of Huanglongbing, which is currently considered the most destructive disease of citrus worldwide. DcRV was previously identified based on metagenomics surveys for virus discovery. Here, we found that this novel and persistent insect reovirus took advantage of a virus-encoded nonstructural protein, P10, for efficient vertical transmission from parents to progeny. P10 assembled into a virion-packaging tubular structure and was associated with oocytes of female D. citri and sperm of males. Consistent with this, knockdown of P10 for either male or female D. citri insects inhibited DcRV transmission to offspring. This tubular strategy for viral spread and biparental transmission might serve as a target for controlling viral vertical transmission and population expansion.

Author(s): Godfrey, Kris; Zalom, Frank; Chiu, Joanna | Abstract: The South American tomato leafminer Tuta Absoluta is a serious and devastating pest of both fresh market and processing tomatoes. Tuta continues to spread throughout the tomato-growing areas of the world. Computer models have predicted that this pest has a moderate likelihood of establishing itself in the commercial tomato producing areas of California, Arizona, and the southern United States. This publication explains the host plants, life cycle, damage of the pest along with monitoring an management methods.

This study presents an economic analysis of cost increases for citrus, avocado and nursery producers should the Diaprepes root weevil become established in California. First identified in Southern California in 2007, Diaprepres would mainly affect orange, grapefruit, lemon and avocado crops. The primary impacts would be increased production costs for pest treatments and increased harvesting costs to conform to quarantine regulations, in particular to ship ornamental plants out of infested regions. The estimated increase in production cost to treat Diaprepes was $609 per acre on average for citrus and avocado and $525 per acre for infested nurseries. The average increase in total cost as a share of revenues was 21.61% for oranges, 11.35% for avocados, 9.80% for grapefruit and 5.62% for lemons; for nursery growers it was less than 1%.

The Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), was discovered as the cause of early decline and death of citrus trees and ornamental plants along coastal Orange and Los Angeles Counties in 2005 and San Diego County in 2006. We established trials to evaluate the effectiveness of two tactics to manage life stages of Diaprepes root weevil that are found in the soil. The first tactic was soil applications of the pesticides bifenthrin or imidacloprid, with and without entomopathogenic nematodes, to kill larvae. The second tactic was attempting to block adult emergence from the soil using a thick wood-chip mulch layer, a small-meshed landscape fabric, and a dry soil barrier (created by subirrigation). We found that soil treatments with the two insecticides (bifenthrin and imidacloprid) at maximum label rates with and without Steinernema riobrave Cabanillas et al. at 5 million nematodes per tree significantly reduced numbers of soil-borne Diaprepes root weevil larvae in one trial compared with the control. Another entomopathogenic nematode, Heterorhabditis indica Poinar et al. applied in wax moth Galleria mellonella L. cadavers at 86 wax moth cadavers per tree was not effective. Adult emergence was reduced by almost 100% when the landscape fabric was kept intact (2009), but emergence occurred in 2010 when the fabric was torn by grove operations (60% reduction). Adult emergence was reduced to ≍70% compared with the control in the subirrigation treatment in 2009 when the summer was hot and dry, but emergence occurred in 2010 during periodic rains toward the end of summer. The mulch treatment kept the soil moist in between irrigations resulting in greater adult emergence rates compared with controls during 2009 and 2010. Health ratings of the trees were taken following several years of physical barrier treatments, and trees treated with the landscape fabric were significantly healthier (mean rating 0.9, scale 0 = healthy to 5 = dead) than those in the other treatments (mean rating of 3.6, 4.0, and 2.5 for control, mulch and subirrigation, respectively). Of the physical barrier methods studied, landscape fabric is most effective in reducing Diaprepes root weevil emergence, increases plant health, and is long lasting, but it is the most costly to install and can be damaged during harvest.

Most of the world’s major citrus production areas were developed outside the citrus centres of origin, separated from many co-evolved natural enemies (pests and pathogens), but progressive globalisation has reunited some pests with their citrus hosts. Additionally, some ‘new-encounter’ pathogens have not co-evolved with citrus. The movement of major citrus pathogens of biosecurity concern is discussed with particular emphasis on tristeza, leprosis, huanglongbing and citrus variegated chlorosis. The chapter details recent attempts to eradicate citrus canker in Florida (USA) and Emerald (Australia) and focusses on the processes and impediments encountered to achieve eradication under very different climatic, legislative and industry conditions. The impact of citrus black spot in areas climatically conducive to the disease and a discussion of fruit as a pathway for introduction of the disease to new areas are discussed. The experience and learning acquired from managing and eradicating these citrus pests will be of value to other countries and regions that are faced with similar pest incursions.

Author(s): Polek, Marylou; Vidalakis, Georgios; Godfrey, Kris | Abstract: Citrus canker and huanglongbing (HLB, or citrus greening) are two serious diseases that affect crops around the world, but have not yet infected California groves. This publication tells how to help keep the diseases out of California, and how to recognize them if or when they do arrive.

Author(s): Haviland, David; Beede, Robert; Godfrey, Kris; Daane, Kent | Abstract: Ferrisia gill is a mealybug species in California that affects pistachio plantings in 11 counties. Find out how to monitor and control the pest's spread using biological and chemical means.

Author(s): Godfrey, Kris; Haviland, David; Erwin, Jeffrey; Daane, Kent; Bentley, Walt | Abstract: Vine mealybug (VMB), a vineyard pest throughout the world, damages grape foliage and fruit, rendering the fruit unmarketable. This publication helps you identify and control VMB in California vineyards.