Unlocking agro-ecosystem sustainability: exploring the bottom-up effects of microbes, plants, and insect herbivores.

Agricultural ecosystem formation and evolution depend on interactions and communication between multiple organisms. Within this context, communication occurs between microbes, plants, and insects, often involving the release and perception of a wide range of chemical cues. Unraveling how this information is coded and interpreted is critical to expanding our understanding of how agricultural ecosystems function in terms of competition and cooperation. Investigations examining dual interactions (e.g. plant-microbe, insect-microbe, and insect-plant) have resolved some basic components of this communication. However, there is a need for systematically examining multitrophic interactions that occur simultaneously between microorganisms, insects, and plants. A more thorough understanding of these multitrophic interactions has been made possible by recent advancements in the study of such ecological interactions, which are based on a variety of contemporary technologies such as artificial intelligence sensors, multi-omics, metabarcoding, and others. Frequently, these developments have led to the discovery of startling examples of each member manipulating the other. Here, we review recent advances in the understanding of bottom-up chemical communication between microorganisms, plants, and insects, and their consequences. We discuss the components of these "chemo-languages" and how they modify outcomes of multi-species interactions across trophic levels. Further, we suggest prospects for translating the current basic understanding of multitrophic interactions into strategies that could be applied in agricultural ecosystems to increase food safety and security.

Response of hibiscus mealybug (Hemiptera: Pseudococcidae) to citrus volatiles induced by mechanical injury.

Hibiscus mealybug, Nipaecoccus viridis (Newstead) (Hemiptera: Pseudococcidae), is a recent invasive pest of citrus and many other crops in Florida. Nipaecoccus viridis attacks all above ground parts of citrus trees and heavy infestation can cause leaf drop and premature abortion of developing fruits. We quantified greater captures of N. viridis in cardboard band traps on areas of citrus trees that were intentionally injured by mechanical rasping of epidermal tissues as compared with similar but uninjured citrus branches. Direct field collection of headspace volatiles from mechanically injured or intact citrus branches revealed both qualitative and quantitative differences. Certain volatiles (γ-terpinene, citronellal, citronellyl acetate, ß-E-farnesene, α-humulene, and α-E-E-farnesene) were only present in samples from damaged citrus branches. Behavioral assays using a laboratory Y-tube olfactometer revealed attraction of N. viridis to volatiles associated with mechanical damage of citrus including synthetic ß-ocimene, γ-terpinene, sabinene, isomers of farnesene, and citronellal when loaded into lures at either of 2 concentrations (0.01 or 0.1 µg/µl). Subsequent field trapping experiments confirmed increased captures of various life stages of N. viridis in cardboard band traps baited with a 10.0 µg/µl concentration of farnesene:ocimene:sabinene blend (in 7:13:17 ratio), as well as those releasing either farnesene or ocimene alone at this same concentration, as compared with the mineral oil (diluent) negative control. Our results indicate that common plant related terpenes released by citrus following mechanical damage may be useful for development of an effective monitoring trap for N. viridis.

Evaluation of diets from various maize hybrids reveals potential tolerance traits against Spodoptera littoralis (Boisd) as measured by developmental and digestive performance.

Spodoptera littoralis (Boisd) (Lepidoptera: Noctuidae) is a highly polyphagous insect that significantly reduces agricultural production of several food staples. We evaluated performance of S. littoralis on several meridic diets based on various maize hybrids, including Oteel, Simon, Valbum, SC703, and SC704. Growth, feeding behaviours, and activity of digestive enzymes of S. littoralis were examined under laboratory conditions. In addition, selected biochemical characteristics of maize hybrid seeds were evaluated, including starch, protein, anthocyanin, as well as phenolic and flavonoid contents, to examine relationships between plant properties and digestive performance of S. littoralis. Performance of S. littoralis on maize hybrids, as measured by nutritional indices, was related to both proteolytic and amylolytic activities quantified using gut extracts. Larval S. littoralis reared on SC703 exhibited the highest efficiency of conversion of digested food, while the lowest was recorded in those fed on the Oteel hybrid. S. littoralis reared on SC703 and Oteel also exhibited the highest and lowest relative growth rates, respectively. The highest levels of proteolytic activity in S. littoralis were measured from larvae reared on the SC703 hybrid, while the lowest levels occurred on the Oteel and Valbum hybrids. Amylolytic activity was lowest in larvae reared on SC703 and Valbum hybrids and highest in larvae reared on the Oteel hybrid. Our results suggest that the SC703 hybrid was the most suitable host for S. littoralis, while the Oteel hybrid demonstrated the greatest level of tolerance against S. littoralis of those evaluated. We discuss the potential utility of maize hybrids exhibiting tolerance traits against this cosmopolitan pest with reference to cultivation of tolerant varieties and identification of specific tolerance traits.

The sandwich feeding assay for use with first instar nymphs of the Asian citrus psyllid, Diaphorina citri confirms the high susceptibility of this life stage to bacterial pesticidal proteins.

Citrus greening or huanglongbing is the most important disease of citrus and threatens citrus production worldwide. As nymphs of Diaphorina citri play a crucial role in the acquisition and transmission of the citrus greening bacterium, suppression of this life stage is particularly important. However, the lack of a tractable feeding assay for use with first instar D. citri nymphs has impeded assessment of the toxicity of bioactives. Of several bacterial pesticidal proteins (BPP) that are toxic to D. citri adults, Mpp51Aa1 and Cry1Ba1, which have LC50 values of 110 and 120 µg/mL respectively in adults, were fed to 1st instar nymphs in a newly developed assay. For this new sandwich feeding assay, parafilm layers containing feeding solution were placed on top of two 35 mm Petri dishes, with a concave surface created on each. Fifty nymphs were transferred to the membrane on one Petri dish, and the second Petri dish placed on the top to create a "sandwich" with the 1st instar nymphs in the middle. Nymphs were fed for four days and the LC50 values for Mpp51Aa1 and Cry1Ba1 were calculated at 6.7 and 41.6 µg/mL respectively. Bioassays with bioengineered plants expressing Cry1Ba1 confirmed that the majority of D. citri mortality occurs during the 1st instar nymph stage, while egg laying adults are much less susceptible. Taken together, these results confirm that 1st instar D. citri nymphs are more susceptible to BPP than adults and demonstrate the utility of the sandwich feeding assay for effective screening of BPPs prior to investment into production of transgenic plants.

Citrus-mediated gene silencing of cytochrome P450 suppresses insecticide resistance and increases mortality in Diaphorina citri.

BACKGROUND: Asian citrus psyllid, Diaphorina citri, is a hemipteran that vectors the causal pathogen of citrus greening disease, or huanglongbing (HLB). HLB is a tree killing disease that has severely limited citrus production globally. Unfortunately, there is no cure for this disease, and mitigation depends on multiple insecticide applications to reduce vector populations. Silencing of cytochrome P450 expression associated with detoxification enzymes by RNA interference is known to increase susceptibility of D. citri to insecticides. However, dsRNA was previously introduced into psyllids by topical applications. The possible application of this technology for pest management will require effective field delivery of the dsRNA. Therefore, we evaluated a virus vector (Citrus tristeza virus; 'mild strain' T36) to deliver gene silencing directly to this sap-sucking insect via plant phloem. Citrus macrophylla plants inoculated with CTV expressing a truncated consensus sequence of CYP450 (CTV-tCYP450) constantly produced small interfering RNA in the plant phloem that targeted five cytochrome p540 (CYP450) genes in D. citri. RESULTS: Insecticide susceptible D. citri reared on citrus infected with CTV-tCYP450 were subsequently more susceptible to imidacloprid, fenpropathrin, carbaryl, and chlorpyrifos than those reared on citrus infected with wildtype CTV or non-infected negative controls. Additionally, nymph survival and adult lifespan were significantly reduced when psyllids were reared on CTV-tCYP450 citrus plants compared with controls. Interestingly, similar results were obtained after one and two generations of rearing. Finally, field-collected psyllids from areas with known broad-spectrum insecticide resistance were rendered more susceptible to imidacloprid and fenpropathrin after feeding on CTV-tCYP450 citrus trees as compared with those reared on controls. CONCLUSION: The integration of citrus-mediated RNA inference targeting psyllid detoxification enzymes could function as a resistance management tool and reduce insecticide input in an integrated pest management program for HLB. © 2024 Society of Chemical Industry.

Reduction of Wolbachia in Diaphorina citri (Hemiptera: Liviidae) increases phytopathogen acquisition and decreases fitness.

Wolbachia pipientis is a maternally inherited intracellular bacterium that infects a wide range of arthropods. Wolbachia can have a significant impact on host biology and development, often due to its effects on reproduction. We investigated Wolbachia-mediated effects in the Asian citrus psyllid, Diaphorina citri Kuwayama, which transmits Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease. Diaphorina citri are naturally infected with Wolbachia; therefore, investigating Wolbachia-mediated effects on D. citri fitness and CLas transmission required artificial reduction of this endosymbiont with the application of doxycycline. Doxycycline treatment of psyllids reduced Wolbachia infection by approximately 60% in both male and female D. citri. Psyllids treated with doxycycline exhibited higher CLas acquisition in both adults and nymphs as compared with negative controls. In addition, doxycycline-treated psyllids exhibited decreased fitness as measured by reduced egg and nymph production as well as adult emergence as compared with control lines without the doxycycline treatment. Our results indicate that Wolbachia benefits D. citri by improving fitness and potentially competes with CLas by interfering with phytopathogen acquisition. Targeted manipulation of endosymbionts in this phytopathogen vector may yield disease management tools.

Bridging biotremology and chemical ecology: a new terminology.

Living organisms use both chemical and mechanical stimuli to survive in their environment. Substrate-borne vibrations play a significant role in mediating behaviors in animals and inducing physiological responses in plants, leading to the emergence of the discipline of biotremology. Biotremology is experiencing rapid growth both in fundamental research and in applications like pest control, drawing attention from diverse audiences. As parallels with concepts and approaches in chemical ecology emerge, there is a pressing need for a shared standardized vocabulary in the area of overlap for mutual understanding. In this article, we propose an updated set of terms in biotremology rooted in chemical ecology, using the suffix '-done' derived from the classic Greek word 'δονέω' (pronounced 'doneo'), meaning 'to shake'.

Spatial and temporal distribution of Scirtothrips dorsalis (Thysanoptera: Thripidae) and their natural enemies in Florida strawberry fields.

Given the recent invasion of Scirtothrips dorsalis Hood in North America, there is limited information regarding their distribution and population dynamics in cultivated small fruit crops. Therefore, we investigated the spatial and temporal distribution of S. dorsalis and their natural enemies in commercially produced strawberry fields in Florida. During 2 consecutive strawberry production seasons, 4 and 6 geographically separated strawberry fields were sampled and were divided into grids with 30-40 sampling points per field. At each sampling point, 4-5 leaf and flower samples were collected, and sticky traps were deployed. We quantified the occurrence of S. dorsalis as well as potential natural enemies, including Orius spp., Geocoris spp., and other predators such as long-legged flies. During both years, most of the S. dorsalis and natural enemies were found on field borders, and counts progressively diminished further into the interiors of plots and away from field edges. Cluster and outlier analysis revealed that S. dorsalis formed statistically significant clusters and that these "hot spots" remained in the same general locations throughout the season. There was a strong relationship between the occurrence of natural enemies and the presence of S. dorsalis, but the number of natural enemies was generally low compared to S. dorsalis. Our results indicate that targeting field borders for chemical control or planting strawberries away from natural areas containing potential alternative hosts for thrips may be an effective strategy for reducing agricultural inputs; however, future field assessments are needed to determine if these methods could replace the treatment of entire fields.

Feeding behavior and hormoligosis associated with imidacloprid resistance in Asian citrus psyllid, Diaphorina citri.

Imidacloprid is a neonicotinoid insecticide used for managing the Asian citrus psyllid, Diaphorina citri Kuwayama, which serves as vector of phytopathogens causing citrus greening. However, development of resistance to neonicotinoids among populations of D. citri has coincided with occasional control failures in the field. The objectives of this research were to (1) survey current levels of imidacloprid resistance in Florida citrus; (2) compare feeding behavior between imidacloprid-resistant and susceptible D. citri using electrical penetration graph recordings, and (3) investigate the possible amplification of insecticide hormoligosis associated with resistance. Field surveys confirmed that the susceptibility of D. citri populations to imidacloprid has decreased in commercial Florida citrus groves compared with a laboratory-susceptible population. Following 12 generations of selection, resistance to imidacloprid increased by 438 fold compared with the susceptible strain. Imidacloprid-susceptible D. citri feeding on citrus exhibited significantly more bouts associated with intercellular pathway (C), phloem penetration (D), phloem salivation (E1), and nonprobing (Np) activities than imidacloprid-resistant counterparts. However, there were no differences observed in the frequency or duration of phloem ingestion or xylem feeding between susceptible and resistant D. citri. There was no statistical difference in fecundity between resistant and susceptible strains. However, the fecundity of imidacloprid-susceptible female D. citri treated with a sublethal concentration of imidacloprid (LC25 ) increased significantly compared with controls, while such hormoligosis was less pronounced among imidacloprid-resistant psyllids. Our results suggest that imidacloprid-resistant psyllids may cease feeding sooner than susceptible counterparts following sublethal exposure to this insecticide, indicative of a behavioral resistance mechanism.

Gene silencing of cathepsins B and L using CTV-based, plant-mediated RNAi interferes with ovarial development in Asian citrus psyllid (ACP), Diaphorina citri.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D. citri females as a result of knockdown of DCcathB and DCcathL genes mediated by CTV dsRNA. These results showed that Psyllids fed on plants treated with the CTV-dsRNA exhibited downregulation of the Vg gene, one of the most important genes associated with embryogenic and female development, which was associated with dsRNA-mediated silencing of the two cathepsin genes. Based on our findings, a CTV-based strategy for delivering RNAi via plants that targets DCcathB and DCcathL genes may represent a suitable avenue for development of dsRNA-based tools to manage D. citri that limits the spread of HLB.

Bacterial Pesticidal Protein Mpp51Aa1 Delivered via Transgenic Citrus Severely Impacts the Fecundity of Asian Citrus Psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP) Diaphorina citri vectors the causative agent of citrus greening disease that has the capacity to decimate citrus production. As an alternative and more sustainable approach to manage D. citri than repeated application of chemical insecticides, we investigated the potential use of the bacteria-derived pesticidal protein, Mpp51Aa1, when delivered by transgenic Citrus sinensis cv. Valencia sweet orange or Citrus paradisi cv. Duncan grapefruit. Following confirmation of transcription and translation of mpp51aa1 by transgenic plants, no impact of Mpp51Aa1 expression was seen on D. citri host plant choice between transgenic and control Duncan grapefruit plants. A slight but significant drop in survival of adult psyllids fed on these transgenic plants was noted relative to those fed on control plants. In line with this result, damage to the gut epithelium consistent with that caused by pore-forming proteins was only observed in a minority of adult D. citri fed on the transgenic Duncan grapefruit. However, greater impacts were observed on nymphs than on adults, with a 40% drop in the survival of nymphs fed on transgenic Duncan grapefruit relative to those fed on control plants. For Valencia sweet orange, a 70% decrease in the number of eggs laid by adult D. citri on transgenic plants was noted relative to those on control plants, with a 90% drop in emergence of progeny. These impacts that contrast with those associated with other bacterial pesticidal proteins and the potential for use of Mpp51Aa1-expressing transgenic plants for suppression of D. citri populations are discussed. IMPORTANCE Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for management of agricultural insect pests and provide a sustainable alternative to the application of chemical insecticides. However, relatively few bacterial pesticidal proteins have been used for suppression of hemipteran or sap-sucking insects such as the Asian citrus psyllid, Diaphorina citri. This insect is particularly important as the vector of the causative agent of citrus greening, or huanglongbing disease, which severely impacts global citrus production. In this study, we investigated the potential of transgenic citrus plants that produce the pesticidal protein Mpp51Aa1. While adult psyllid mortality on transgenic plants was modest, the reduced number of eggs laid by exposed adults and the decreased survival of progeny was such that psyllid populations dropped by more than 90%. These results provide valuable insight for potential deployment of Mpp51Aa1 in combination with other control agents for the management of D. citri.

Laboratory evaluation of the life history of hibiscus mealybug, Nipaecocus viridis (Hemiptera: Pseudococcidae), on selected citrus and potential non-citrus hosts in Florida.

Nipaecoccus viridis (Newstead) (Hemiptera: Pseudococcidae) is an emerging invasive pest of citrus. There is no information on the life history of N. viridis in relation to citrus cultivars produced in Florida. Here, we quantified the life-history parameters of N. viridis on 6 citrus and 3 non-citrus host plant species under laboratory conditions. We also investigated the effect of mode of reproduction (sexual vs. parthenogenic) on the fecundity of N. viridis. Nipaecoccus viridis was able to develop to maturity and reproduce on all host plant taxa tested. The individual host plants affected the developmental and survival rate, adult longevity, and offspring production of N. viridis. All citrus genotypes commercially grown in Florida were susceptible to N. viridis, with the exception of Citrus aurantium L., which suggests that there is need for management of this invasive pest in Florida citrus groves. Murraya paniculata L., Hibiscus rosa-sinensis L., and Punica granatum L. were also acceptable hosts for N. viridis. Sexually reproducing female N. viridis produced significantly more eggs than those reproducing asexually. Based on our results, propagation of M. paniculata and other citrus relatives should be discouraged around citrus groves in Florida. Given the fitness benefit of sexual reproduction in this pest, pheromone-based tools like mating disruption and mass trapping of adult males could be considered as part of an integrated pest management program with insecticides.

Inoculation of Tomato With Plant Growth Promoting Rhizobacteria Affects the Tomato-Potato Psyllid-Candidatus Liberibacter Solanacearum Interactions.

The Rio Grande Valley (RGV) in southern Texas is well-suited for vegetable production due to its relatively mild/warm weather conditions in the fall and winter. Consequently, insects inflict year-round, persistent damage to crops in the RGV and regions with similar climate. Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), commonly known as the potato psyllid, is a known vector of Candidatus Liberibacter solanacearum (CLso) (Hyphomicrobiales: Rhizobiaceae), a fastidious phloem-limited bacterium associated to vein-greening in tomatoes and Zebra Chip in potatoes. Vector control is the primary approach of integrated pest management (IPM) strategies that aim to prevent plant diseases in commercial agricultural systems. However, resistance-selective pressures that decrease the effectiveness of chemical control (insecticide) applications over time are of increasing concern. Therefore, we explore an ecological approach to devising alternative IPM methodologies to manage the psyllid-transmitted CLso pathogen to supplement existing chemical products and application schedules without increasing resistance. In this study, our objective was to examine the effects of plant-growth promoting rhizobacteria (PGPR) on host-vector-pathogen interactions. Soil-drench applications of PGPRs to Solanum lycopersicum (Solanales: Solanaceae) seedlings revealed structural and possible physiological changes to the plant host and indirect changes on psyllid behavior: host plants had increased length and biomass of roots and exhibited delayed colonization by CLso, while psyllids displayed changes in parental (F0) psyllid behavior (orientation and oviposition) in response to treated hosts and in the sex ratio of their progeny (F1). Based on our results, we suggest that PGPR may have practical use in commercial tomato production.

Foliar Antibiotic Treatment Reduces Candidatus Liberibacter asiaticus Acquisition by the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae), but Does not Reduce Tree Infection Rate.

Huanglongbing (HLB), or citrus greening, is the most destructive disease of cultivated citrus worldwide. Candidatus Liberibacter asiaticus (CLas), the putative causal agent of HLB, is transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). In Florida, D. citri was first reported in 1998, and CLas was confirmed in 2005. Management of HLB relies on the use of insecticides to reduce vector populations. In 2016, antibiotics were approved to manage CLas infection in citrus. Diaphorina citri is host to several bacterial endosymbionts and reducing endosymbiont abundance is known to cause a corresponding reduction in host fitness. We hypothesized that applications of oxytetracycline and streptomycin would reduce: CLas populations in young and mature citrus trees, CLas acquisition by D. citri, and D. citri abundance. Our results indicate that treatment of citrus with oxytetracycline and streptomycin reduced acquisition of CLas by D. citri adults and emerging F1 nymphs as compared with that observed in trees treated only with insecticides, but not with antibiotics. However, under field conditions, neither antibiotic treatment frequency tested affected CLas infection of young or mature trees as compared with insecticide treatment alone (negative control); whereas trees enveloped with mesh screening that excluded vectors did prevent bacterial infection (positive control). Populations of D. citri were not consistently affected by antibiotic treatment under field conditions, as compared with an insecticide only comparison. Collectively, our results suggest that while foliar application of oxytetracycline and streptomycin to citrus reduces acquisition of CLas bacteria by the vector, even high frequency applications of these formulations under field conditions do not prevent or reduce tree infection.

Cry1Ba1-mediated toxicity of transgenic Bergera koenigii and Citrus sinensis to the Asian citrus psyllid Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus greening disease, which has severely impacted citrus production on a global scale. As the current repeated application of chemical insecticides is unsustainable for management of this insect and subsequent protection of groves, we investigated the potential use of the bacteria-derived pesticidal protein, Cry1Ba1, when delivered via transgenic citrus plants. Having demonstrated transformation of the Indian curry leaf tree, Bergera koenigii, for Cry1Ba1 expression for use as a trap plant, we produced transgenic plants of Duncan grapefruit, Citrus paridisi, Valencia sweet orange, Citrus sinensis, and Carrizo citrange, C. sinensis x Poncirus trifoliata, for expression of Cry1Ba1. The presence of the cry1ba1 gene, and cry1ba1 transcription were confirmed. Western blot detection of Cry1Ba1 was confirmed in most cases. When compared to those from wild-type plants, leaf discs from transgenic Duncan and Valencia expressing Cry1Ba1 exhibited a "delayed senescence" phenotype, similar to observations made for transgenic B. koenigii. In bioassays, significant reductions in the survival of adult psyllids were noted on transgenic B. koenigii and Valencia sweet orange plants expressing Cry1Ba1, but not on transgenic Duncan grapefruit or Carrizo citrange. In contrast to psyllids fed on wild type plants, the gut epithelium of psyllids fed on transgenic plants was damaged, consistent with the mode of action of Cry1Ba1. These results indicate that the transgenic expression of a bacterial pesticidal protein in B. koenigii and Valencia sweet orange offers a viable option for management of D. citri, that may contribute to solutions that counter citrus greening disease.

Life Table Parameters and Digestive Enzyme Activity of Spodoptera littoralis (Boisd) (Lepidoptera: Noctuidae) on Selected Legume Cultivars.

Spodoptera littoralis (Boisd) is a highly destructive pest that attacks a large number of economically important crops. We examined life table parameters as well as activity of major digestive enzymes of S. littoralis larvae in response to protein and starch contents across 11 legume cultivars to identify potential resistance traits. The results showed that S. littoralis reared on the common bean, Arabi, displayed the highest intrinsic rate of increase (r), while the lowest was recorded on the cowpea, Mashhad. Also, the highest net reproductive rate (R0) was obtained in those insects reared on the Arabi cultivar. Larvae displayed the highest and lowest proteolytic activities when fed on Mashhad and Arabi cultivars, respectively. The highest amylolytic activity was quantified in larvae that fed on the Arabi and 1057 cultivars, while the lowest occurred in larvae feeding on Yaghout and Mashhad cultivars. Developmental time of S. littoralis larvae was negatively correlated with protein content, while amylolytic activity was positively correlated with starch content of legumes. Our findings indicate that Arabi was a susceptible cultivar, while Mashhad exhibited tolerance traits against S. littoralis. These results should facilitate selection of legume cultivars for production or breeding efforts that involve S. littoralis management.

Genetic Modification of Bergera koenigii for Expression of the Bacterial Pesticidal Protein Cry1Ba1.

The curry leaf tree, Bergera koenigii, is highly attractive to the Asian citrus psyllid, Diaphorina citri, which vectors the bacterial causative agent of citrus greening or huanglongbing disease. This disease has decimated citrus production in Florida and in other citrus-producing countries. As D. citri exhibits high affinity for feeding on young leaves of B. koenigii, transgenic B. koenigii expressing bacteria-derived pesticidal proteins such as Cry1Ba1 have potential for D. citri management when planted in or adjacent to citrus groves. Importantly, the plant pathogenic bacterium that causes citrus greening does not replicate in B. koenigii. Transgenic plants of B. koenigii were produced by insertion of the gene encoding the active core of the pesticidal protein Cry1Ba1 derived from Bacillus thuringiensis. The transformation success rate was low relative to that of other citrus, at 0.89%. T-DNA integration into the genome and cry1ba1 transcription in transgenic plants were confirmed. Transgenic plants expressing Cry1Ba1 differed from wild-type plants, differed in photosynthesis parameters and hormone levels in some instances, and a marked delay in wilting of detached leaves. The gut epithelium of D. citri fed on transgenic plants was severely damaged, consistent with Cry1Ba1-mediated pore formation, confirming expression of the pesticidal protein by transgenic B. koenigii. These results demonstrate that transgenic B. koenigii expressing bacteria-derived pesticidal proteins can be produced for potential use as trap plants for suppression of D. citri populations toward protection of citrus groves from citrus greening.

Detectability of Hibiscus Mealybug, Nipaecoccus viridis (Hemiptera: Pseudoccocidae), DNA in the Mealybug Destroyer, Cryptolaemus montrouzieri (Coleoptera: Coccinellidae), and Survey of Its Predators in Florida Citrus Groves.

The Hibiscus mealybug, Nipaecoccus viridis (Newstead), has recently established in Florida citrus and become a pest of concern given secondary pest outbreaks associated with management of citrus greening disease. Chemical controls used to manage other citrus arthropod pests are not as effective against N. viridis due to its waxy secretions, clumping behavior, and induced cellular changes to host plant tissue which increase microhabitats. Populations of this mealybug pest are regulated by natural enemies in its native region, but it remains unclear if resident natural enemies in Florida citrus could similarly suppress N. viridis populations. This investigation: 1) established species-specific primers for N. viridis based on the mitochondrial gene Cytochrome-oxidase 1 (COI), 2) determined duration of N. viridis DNA detectability in a known predator, the mealybug destroyer (Cryptolaemus montrouzieri Mulsant), by using identified primers in molecular gut content analysis, and 3) screened field-collected predators for the presence of N. viridis DNA. The detection rate of N. viridis DNA was >50% at 36 h after adult C. montrouzieri feeding but DNA was no longer detectable by 72 h after feeding. Field-collected predators were largely comprised of spiders, lacewings, and C. montrouzieri. Spiders, beetles (primarily C. montrouzieri), and juvenile lacewings were the most abundant predators of N. viridis, with 17.8, 43.5, and 58.3 of field-collected samples testing positive for N. viridis DNA, respectively. Our results indicate that Florida citrus groves are hosts to abundant predators of N. viridis and encourage the incorporation of conservation or augmentative biological control for management of this pest.

Salicylic acid mediated immune response of Citrus sinensis to varying frequencies of herbivory and pathogen inoculation.

BACKGROUND: Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CaLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. RESULTS: A one-time (7 days) inoculation access period with CaLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response after a long period of infection without herbivory (270 and 360 days). In contrast, while repeated (monthly) 'pulses' of 7 day feeding injury by psyllids stimulated immunity in CaLas-infected citrus by increasing SA in leaves initially (up to 120 days), long-term (270 and 360 days) repeated herbivory caused SA to decrease coincident with upregulation of genes associated with SA metabolism (BMST and DMR6). Similarly, transcriptional responses and metabolite (SA and its analytes) accumulation in citrus leaves exposed to a continuously reproducing population of D. citri exhibited a transitory upregulation of genes associated with SA signaling at 120 days and a posterior downregulation after long-term psyllid (adults and nymphs) feeding (270 and 360 days). CONCLUSIONS: Herbivory played an important role in regulation of SA accumulation in mature leaves of C. sinensis, whether or not those trees were coincidentally infected with CaLas. Our results indicate that prevention of feeding injury inflicted by D. citri from the tritrophic interaction may allow citrus plants to better cope with the consequences of CaLas infection, highlighting the importance of vector suppression as a component of managing this cosmopolitan disease.

Wind Speed and Direction Drive Assisted Dispersal of Asian Citrus Psyllid.

Wind directly influences the spread of vector-borne plant pathogens by driving the passive dispersal of vectors to potentially new areas. Here, we evaluated the effect of wind speed and direction on the dispersal of the Asian citrus psyllid (ACP), Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae), the vector of the bacteria causing huanglongbing (HLB), a lethal disease of citrus. The effect of different wind speeds on short or long-distance dispersal of ACP was investigated using a high-speed wind tunnel under laboratory conditions. The effect of wind direction on ACP dispersal under field conditions was evaluated using custom-made wind vane-style traps. In wind tunnel assays, ACP remained on plants until wind treatments reached ≥48 km/h when psyllids were mostly dislodged from plants and moved by the wind. For a short-distance, wind-driven movement (movement by the wind from one plant to another), the effect of wind speed was not significant at any of the wind speed treatments tested. Wind vane traps placed in a Florida citrus grove captured significantly more ACP on the windward side, suggesting that ACP were moved with the wind. The number of ACP found on the windward side of traps was significantly higher from May to August. These results indicate that ACP is likely to disperse with prevailing wind direction and that settled ACP may become dislodged and moved at random by high wind speeds occurring in areas of significant citrus production (southern California, Florida, or Texas).