Role of chitinase expression in the virulence of Lecanicillium lecanii against citrus black aphid (Toxoptera aurantii).

Chitinase plays a vital role in the virulence of entomopathogenic fungi (EPF) when it infects host insects. We used gene recombination technology to express chitinase of three strains of Lecanicillium lecanii: Vl6063, V3450, and Vp28. The ORF of ChitVl6063, ChitV3450 and ChitVp28 were inserted into the fungal expression vector pBARGPE-1, which contained strong promoter and terminator, respectively, to construct a chitinase overpressing plasmid, then transformed the wild-type strain with blastospore transformation method. The virulence of the three recombinant strains against Toxoptera aurantii was improved by overproduction of ChitVl6063, ChitV3450, and ChitVp28, as demonstrated by significantly lower 3.43 %, 1.72 %, and 1.23 % fatal doses, respectively, according to an insect bioassay. Similarly, lethal times of recombinants (ChitVl6063, ChitV3450 and ChitVp28) were also decreased up to 29.51 %, 30.46 % and 33.90 %, respectively, compared to the wild-type strains. Improving the expression of chitinase is considered as an effective method for the enhancement of the EPF value. The efficacy could be enhanced using recombinant technology, which provides a prospecting view for future insecticidal applications.

Demography and Population Projection of Tetranychus urticae (Tetranychidae) on Phaseolus vulgaris (Fabaceae) Colonized by Entomopathogenic Fungal Endophytes.

Tetranychus urticae is a highly polyphagous and global pest. Spider mites primarily feed on the underside of leaves, resulting in decreased photosynthesis, nutritional loss, and the development of chlorotic patches. We investigated the life tables of the two-spotted spider mite T. urticae on fungal endophyte Beauveria bassiana colonized and untreated plants of the common Phaseolus vulgaris L., a bean plant. Based on the age-stage, two-sex life table theory, data were evaluated. The mites raised on untreated plants had protonymphs, deutonymphs, and total pre-adult stage durations that were considerably shorter (1.76, 2.14, and 9.77 d, respectively) than the mites raised on plants that had been colonized (2.02, 2.45, and 10.49 d, respectively). The fecundity (F) varied from 28.01 eggs per female of colonized plants to 57.67 eggs per female of endophyte-untreated plants. The net reproductive rate (R0) in the plants with and without endophytes was 19.26 and 42.53 brood, respectively. The untreated plants had an intrinsic rate of increase (rm) of 0.245 days as opposed to the colonized plants, which had an r of 0.196 days and a finite rate of increase (λ) (1.27 and 1.21, respectively). Population forecasts based on a two-sex, age-stage life table demonstrated the dynamism and variability of the stage structure. Furthermore, the colonization of B. bassiana had a negative impact on the growth and development of T. urticae. It lowered the adult mite life span, female fecundity, net reproduction rate, and intrinsic growth rate. We propose that future research should better use entomopathogenic fungal endophytes to understand host plant resistance strategies in integrated pest management.

Genome-wide identification and immune response analysis of mitogen-activated protein kinase cascades in tea geometrid, Ectropis grisescens Warren (Geometridae, Lepidoptera).

BACKGROUND: Tea geometrid Ectropis grisescens (Geometridae: Lepidoptera), is one of the most destructive defoliators in tea plantations in China. The MAPK cascade is known to be an evolutionarily conserved signaling module, acting as pivotal cores of host-pathogen interactions. Although the chromosome-level reference genome of E. grisescens was published, the whole MAPK cascade gene family has not been fully identified yet, especially the expression patterns of MAPK cascade gene family members upon an ecological biopesticide, Metarhizium anisopliae, remains to be understood. RESULTS: In this study, we have identified 19 MAPK cascade gene family members in E. grisescens, including 5 MAPKs, 4 MAP2Ks, 8 MAP3Ks, and 2 MAP4Ks. The molecular evolution characteristics of the whole Eg-MAPK cascade gene family, including gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication, were systematically investigated. Our results showed that the members of Eg-MAPK cascade gene family were unevenly distributed in 13 chromosomes, and the clustered members in each group shared similar structures of the genes and proteins. Gene expression data revealed that MAPK cascade genes were expressed in all four developmental stages of E. grisescens and were fairly and evenly distributed in four different larva tissues. Importantly, most of the MAPK cascade genes were induced or constitutively expressed upon M. anisopliae infection. CONCLUSIONS: In summary, the present study was one of few studies on MAPK cascade gene in E. grisescens. The characterization and expression profiles of Eg-MAPK cascades genes might help develop new ecofriendly biological insecticides to protect tea trees.

Comprehensive genome-wide identification and expression profiling of ADF gene family in Citrus sinensis, induced by endophytic colonization of Beauveria bassiana.

Endophytic entomopathogenic species are known to systematically colonize host plants and form symbiotic associations that benefit the plants they live with. The actin-depolymerizing factors (ADFs) are a group of gene family that regulate growth, development, and defense-related functions in plants. Systematic studies of ADF family at the genome-wide level and their expression in response to endophytic colonization are essential to understand its functions but are currently lacking in this field. 14ADF genes were identified and characterized in the Citrus sinensis genome. The ADF genes of C. sinensis were classified into five groups according to the phylogenetic analysis of plant ADFs. Additionally, the cis-acting analysis revealed that these genes play essential role in plant growth/development, phytohormone, and biotic and abiotic responses; and the expression analysis showed that the symbiotic interactions generate a significant expression regulation level of ADF genes in leaves, stems and roots, compared to controls; thus enhancing seedlings' growth. Additionally, the 3D structures of the ADF domain were highly conserved during evolution. These results will be helpful for further functional validation of ADFs candidate genes and provide important insights into the vegetative growth, development and stress tolerance of C. sinensis in responses to endophytic colonization by B. bassiana.

Genome-Wide Identification and Characterization of Toll-like Receptors (TLRs) in Diaphorina citri and Their Expression Patterns Induced by the Endophyte Beauveria bassiana.

Toll-like receptors (TLRs) are pathogen recognition receptors (PRRs), which play key roles in helping the host immune system fight pathogen invasions. Systematic information on TLRs at the genome-wide level and expression profiling in response to endophytic colonization is very important to understand their functions but is currently lacking in this field. Here, a total of two TLR genes were identified and characterized in Diaphorina citri. The TLR genes of D. citri were clustered into five families according to the phylogenetic analysis of different species' TLRs. The domain organization analyses suggested that the TLRs were constituted of three important parts: a leucine-rich repeat (LRR) domain, a transmembrane region (TR) and a Toll/interleukin-1 receptor (TIR) domain. The mRNA expression levels of the two TLR genes (DcTOLL and DcTLR7) were highly regulated in both nymphs and adults of D. citri. These results elucidated the potentiated TLR gene expression in response to endophytically colonized plants. Furthermore, the 3D structures of the TIR domain were highly conserved during evolution. Collectively, these findings elucidate the crucial roles of TLRs in the immune response of D. citri to entomopathogens systematically established as endophytes, and provide fundamental knowledge for further understanding of the innate immunity of D. citri.

Comparative microbiome analysis of Diaphorina citri and its associated parasitoids Tamarixia radiata and Diaphorencyrtus aligarhensis reveals Wolbachia as a dominant endosymbiont.

Microbiome analysis in a host-parasitoid interaction network was conducted to compare the taxonomic composition of bacterial communities of Diaphornia citri, Tamarixia radiata, and Diaphorencyrtus aligarhensis. The comparative analysis revealed differences in the composition and diversity of the symbiont populations across the host and its associated parasitoids. Proteobacteria was the most dominant phylum, representing 67.80% of the total bacterial community, while Candidatus Profftella armature and Wolbachia were the dominant genera across the host and parasitoids. There were clear differences observed in alpha and beta diversity of microbiota through the host and its associated parasitoids. The function prediction of bacterial communities and Pearson correlation analysis showed that specific bacterial communities displayed positive correlations with the carbohydrate metabolism pathway. Furthermore, when symbiotic bacteria were eliminated using a broad-spectrum antibiotic, tetracycline hydrochloride, the parasitoids' median survival time and longevity were significantly reduced. We confirmed the physiological effects of symbiotic bacteria on the fitness of parasitoids and demonstrated the effect of antibiotics in decreasing the food intake and measurement of amino acids in the hemolymph. This study sheds light on basic information about the mutualism between parasitoids and bacteria, which may be a potential source for biocontrol strategies for citrus psyllid, especially D. citri.

Comparative transcriptome analysis of Tamarixia radiata (Hymenoptera: Eulophidae) reveals differentially expressed genes upon heat shock.

Temperature is a key parameter that affects insect population, abundance, and distribution in tropical and subtropical regions. Tamarixia radiata Waterson (Hymenoptera: Eulophidae) is a species-specific ectoparasitoid widely used as a biological control agent for the major citrus pest Diphornia citri Kuwayama (Hemiptera: Liviidea). To date, T. radiata response to high temperature at the molecular level still is unclear. In this study, we conducted a comparative analysis of the transcriptomes of T. radiata exposed at 25 °C and 38 °C for 15 min. A total of 51,072 unigenes were obtained, 22,413 annotated with a mean length of 1054 bp. Differential expression analysis showed that 502 genes were identified, including 476 genes significantly up-regulated and 26 genes down-regulated after heat stress exposure. The Gene Ontology analysis showed that most enriched DEGs are categorized into "cellular process", "metabolic process" and "DNA binding." In addition, "Lysosome," "Longevity regulating pathway-multiple species," and "starch and sucrose metabolism" were highly enriched in Kyoto Encyclopedia of Genes and Genomes pathways. Transcriptome analyses showed that heat stress significantly induced the transcription of the molecular chaperone, immune response, stress signaling transduction, and oxidation resistance, including highly expressed heat shock proteins, ATPases, and detoxifying enzymes. Furthermore, the expression patterns of thirteen genes including heat shock proteins (HSP), glutathione S-transferase (GST) and cytochrome P450 were consistent with the transcriptome results obtained through qRT-PCR. Together, our results provided a comprehensive study of the molecular response of T. radiata to heat stress and provides new insight for the future functional validation of heat resistance-related genes.

Temperature-Dependent Biological Control Effectiveness of Tamarixia radiata (Hymenoptera: Eulophidea) Under Laboratory Conditions.

The parasitism rate and host-feeding rate of Tamarixia radiata (Hymenoptera: Eulophidae), an ectoparasitoid of Diaphorina citri (Hemiptera: Liviidae), were evaluated at 20, 27.5, 30, and 35°C, at 70 ± 5% RH, and 14 h of photoperiod. The biological control efficacy of T. radiata was evaluated by linking the age-stage predation rate with the two-sex life table. The net host-feeding rate (C0) by T. radiata was 32.05, 54.40, 17.25, and 1.92 nymphs per female parasitoid at 20, 27.5, 30, and 35°C, respectively. The total net nymphs killing rate (Z0) was 103.02, 223.82, 72.95, and 6.60 nymphs per female parasitoid at 20, 27.5, 30, and 35°C, respectively. Noneffective parasitism rate was observed at 35°C because of high mortality at this temperature. Our results indicated that temperature had meaningful effects on parasitism and host-feeding rate parameters in the laboratory, and may affect biological control efficiency of the parasitoid in the field. The highest host-feeding rate and total biological control efficiency of T. radiata were recorded at 27.5°C. Most importantly, we found that host-feeding activity of the parasitoid is temperature-dependent, and changed across temperature regimes: the host-feeding rate increased as the temperature increased up to 30°C, started to decrease after this temperature and declined to its minimum level at 35°C. This information is valuable for developing biological control and integrated pest management techniques for Asian citrus psyllid management.

Host-pathogen interaction between Asian citrus psyllid and entomopathogenic fungus (Cordyceps fumosorosea) is regulated by modulations in gene expression, enzymatic activity and HLB-bacterial population of the host.

The host-pathogen interaction has been explored by several investigations, but the impact of fungal pathogens against insect resistance is still ambiguous. Therefore, we assessed the enzymatic activity and defense-related gene expression of Asian citrus psyllid (ACP) nymphal and adult populations on Huanglongbing-diseased citrus plants under the attack of Cordyceps fumosorosea. Overall, five enzymes viz. superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione S-transferase (GST), carboxylesterase (CarE), and four genes, namely SOD, 16S, CYP4C68, CYP4BD1, were selected for respective observations from ACP populations. Enzymatic activity of four enzymes (SOD, POD, GST, CarE) was significantly decreased after 5-days post-treatment (dpt) and 3-dpt fungal exposure in fungal treated ACP adult and nymphal populations, respectively, whereas the activity of CAT was boosted substantially post-treatment time schedule. Besides, we recorded drastic fluctuations in the expression of CYP4 genes among fungal treated ACP populations. After 24 hours post-treatment (hpt), expression of both CYP4 genes was boosted in fungal treated populations than controlled populations (adult and nymph). After 3-dpt, however, the expression of CYP4 genes was declined in the given populations. Likewise, fungal attack deteriorated the resistance of adult and nymphal of ACP population, as SOD expression was down-regulated in fungal-treated adult and nymphs after 5-dpt and 3-dpt exposure, respectively. Moreover, bacterial expression via the 16S gene was significantly increased in fungal-treated adult and nymphal ACP populations with increasing post-treatment time. Overall, our data illustrate that the fungal application disrupted the insect defense system. The expression of these genes and enzymes suppress the immune function of adult and nymphal ACP populations. As it is reported first time that the applications of C. fumosorosea against ACP reduce insect resistance by interfering with the CYP4 and SOD system. Therefore, we propose new strategies to discover the role of certain toxic compounds from fungus, which can reduce insect resistance, focusing on resistance-related genes and enzymes.

Diamondback Moth Larvae Trigger Host Plant Volatiles that Lure Its Adult Females for Oviposition.

The diamondback moth (DBM) is a destructive pest of crucifer crops. In this study, DBM larvae shown to herbivore induced plant volatiles (HIPVs) that were attractive to adult females exposed in a Y-tube olfactometer. Our results showed that olfactory responses of adult females to HIPVs induced by third instar larvae feeding on Barbarea vulgaris were significantly higher (20.40 ± 1.78; mean moths (%) ± SD) than those induced by first instar larvae (14.80 ± 1.86; mean moths (%) ± SD). Meanwhile, a significant concentration of Sulphur-containing isothiocyanate, 3-methylsulfinylpropyl isothiocyanate, and 4-methylsulfinyl-3-butenyl isothiocyanate were detected in HIPVs released by third instar larvae compared to those released by first instar larvae while feeding on B. vulgaris. When the DBM females were exposed to synthetic chemicals, singly and in blend form, a similar response was observed as to natural HIPVs. Our study demonstrated that the relationship between isothiocyanates acting as plant defense compounds, host plant cues emission and regulation of the DBM adult female behavior due to key volatile triggered by the DBM larvae feeding on B. vulgaris.

Characterization of mycotoxins from entomopathogenic fungi (Cordyceps fumosorosea) and their toxic effects to the development of asian citrus psyllid reared on healthy and diseased citrus plants.

Entomopathogenic fungi (EPF) produce multiple mycotoxins, which play an essential role in improving fungal pathogenesis and virulence. To characterize various mycotoxins from the crude methanol extract of Cordyceps fumosorosea, a major EPF against various insect pests, we performed ultra-performance liquid chromatography coupled to quadrupole time of flight mass spectrometer (UPLC-QTOF MS) technique, and all compounds were identified through molecular mass and formulae. Bassianolide was assessed against the nymphs and adults of Diaphorina citri reared on healthy and Huánglóngbìng (HLB)-diseased Citrus spp. Plants under laboratory conditions. Overall, 17 compounds were identified from the fungal extract and categorized into three groups, i.e. (1) alkaloids (Isariotins A-C), (2) peptides (Bassianolide, Beauverolides, Beauvericin A, Isaridins and Destruxin E) and (3) polyketide (Tenuipyrone). The detected beauverolides (B, C, F, I, Ja) from C. fumosorosea were novel mycotoxins, and their detection intensity was the highest in the fungal extract. Furthermore, bassianolide caused more than 70% and 80% mortality of D. citri nymphs and adults after two days of application, respectively. After three days of chemical application, all nymphal and adult populations of D. citri were killed by bassianolide. However, the mortality rates of both populations, nymphs and adults, were higher on HLB-diseased plants as compared to healthy plants.

Fungal Endophyte Communities of Crucifer Crops Are Seasonally Dynamic and Structured by Plant Identity, Plant Tissue and Environmental Factors.

Endophytic fungi are important in diverse plant functions but knowledge of the factors that shape assemblages of these symbionts is lacking. Here, using a culture-dependent approach, we report 4,178 endophytic fungal isolates representing 16 orders isolated from stems, roots and leaves of three cruciferous plant species, Chinese cabbage (Brassica rapa L.), radish (Raphanus sativus L.) and white cabbage (B. olerocea L.), collected from 21 focal fields with different landscape contexts and pesticide uses during four seasons (summer, autumn, winter and spring). The colonization rate of fungi was found to be most strongly affected by season, plant identity and plant tissue. The colonization was highest during autumn, followed by summer, spring and lowest during winter. The colonization was highest in B. olerocea (53.2%), followed by B. rapa (42.6%), and lowest in R. sativus (35.0%). The colonization was highest in stems (51.9%) in all plant types, followed by leaves (42.4%) and roots (37.5%). Hypocreales was the dominant order (33.3% of all the isolates), followed by Glomerellales (26.5%), Eurotiales (12.1%), Pleosporales (9.8%) and Capnodiales (6.0%). Fungal endophyte abundance (number of isolates) followed the same pattern as colonization rate, while species richness varied with season and host plant tissue. Ordination analyses showed that the abundance and richness of Hypocreales, Eurotiales and Sordariales were associated with plant roots, while Capnodiales, Pleosporales and Trichosphaeriales were associated with spring. Other environmental factors, elevation, and the proportions of grassland, forest, orchard and waterbodies in the surrounding landscape also exerted effects within some categories of other main effects or for certain fungal taxa. Our results indicate that while fungal endophyte communities of crucifer crops vary strongly with the season, they are also strongly structured by plant identity and plant tissue, to a lesser extent by pesticide use and only weakly by landscape composition. The understanding of the ecological roles of fungal endophytes could contribute to habitat management and consequently improve crop pest management.

Effects of Seedling Age on Colonization Patterns of Citrus limon Plants by Endophytic Beauveria bassiana and Metarhizium anisopliae and Their Influence on Seedlings Growth.

The inoculation methods, the fungal strains, and several other factors are known to influent the success of fungal entomopathogens colonization in plants. The physiological status of the plant could also be another determinant. In the present study, the ability of three strains of Beauveria bassiana and one strain of Metarhizium anisopliae to successfully colonize Citrus limon plants and the influence of seedling age on endophytic colonization success was examined. Three, 4, and 6 months old seedlings were inoculated with 10 mL of 1 × 108 conidial·mL-1 suspensions of each of the four fungal strains via foliar spraying. All fungal strains successfully colonized citrus seedlings and were sustained up to 2 months in colonized plants irrespective of the seedling age, with differences in the mean percentage colonization recorded at various post-inoculation periods among the fungal strains. The highest percent endophytic fungi recovery rate was recorded in the 3 months old seedlings, where fungal mycelia of inoculated fungi were successfully re-isolated from 65.6% of the untreated newly developed leaf and stem tissues. One strain of B. bassiana, BB Fafu-12, significantly improved seedling height and leaf number. The study demonstrates the influence of seedling age on B. bassiana and M. anisopliae successful colonization in the citrus plant.

Temperature-Dependent Demography and Population Projection of Tamarixia radiata (Hymenoptera: Eulophidea) reared on Diaphorina citri (Hemiptera: Liviidae).

Tamarixia radiata Waterson (Hymenoptera: Eulophidea) is the principal natural enemy used for the control of the major citrus pest Diaphorina citri Kuwayama (Hemiptera: Liviidae). In this study, we collected the life-history data of T. radiata at four different temperatures (20, 27.5, 30, and 35°C) and analyzed them by using the age-stage, two-sex life table. The longest preadult developmental time (16.53 d) was observed at 20°C, whereas the shortest one was 7.29 d at 35°C. The preadult development rate was well-fitted to a linear equation. The lower threshold temperature (T0) was 7.85°C and the thermal summation (K) was 193.36 degree-day. The highest fecundity (F) was 322.7 eggs per female was at 27.5°C, whereas the lowest one was 10.8 eggs per female at 35°C. The net reproductive rate (R0) were 70.97, 169.42, 55.70, and 3.25 offspring at 20, 27.5, 30, and 35°C, respectively; the intrinsic rate of increase (r) were 0.1401, 0.3167, 0.3517, and 0.1143 d-1, respectively. The highest values of fecundity, net reproductive rate, intrinsic rate of increase, and finite rate of increase were observed at 27.5°C. The relationships among F, R0, Nf, and N in all treatments were consistent with R0=F×(Nf/N). The age of peak reproductive value was close to the total preoviposition period in all treatments. Population projections based on the age-stage, two-sex life table showed the dynamics of stage structure and its variability. Faster population growth was observed at 27.5 and 30°C.

Specialist and Generalist Fungal Parasites Induce Distinct Biochemical Changes in the Mandible Muscles of Their Host.

Some parasites have evolved the ability to adaptively manipulate host behavior. One notable example is the fungus Ophiocordyceps unilateralis sensu lato, which has evolved the ability to alter the behavior of ants in ways that enable fungal transmission and lifecycle completion. Because host mandibles are affected by the fungi, we focused on understanding changes in the metabolites of muscles during behavioral modification. We used High-Performance Liquid Chromatography-Mass/Mass (HPLC-MS/MS) to detect the metabolite difference between controls and O. unilateralis-infected ants. There was a significant difference between the global metabolome of O. unilateralis-infected ants and healthy ants, while there was no significant difference between the Beauveria bassiana treatment ants group compared to the healthy ants. A total of 31 and 16 of metabolites were putatively identified from comparisons of healthy ants with O. unilateralis-infected ants and comparisons of B. bassiana with O. unilateralis-infected samples, respectively. This result indicates that the concentrations of sugars, purines, ergothioneine, and hypoxanthine were significantly increased in O. unilateralis-infected ants in comparison to healthy ants and B. bassiana-infected ants. This study provides a comprehensive metabolic approach for understanding the interactions, at the level of host muscles, between healthy ants and fungal parasites.

Endophytic Beauveria bassiana in Foliar-Treated Citrus limon Plants Acting as a Growth Suppressor to Three Successive Generations of Diaphorina citri Kuwayama (Hemiptera: Liviidae).

Entomopathogenic fungi are commonly applied as inundative sprays to protect plants against insect pests. Their artificial establishment as fungal endophytes to provide other benefits to the host plants aside mere protection against the primary pests has also been widely demonstrated. In the present study, two fungal strains of Beauveria bassiana and one strain of Isaria fumosorosea were assessed in a pathogenicity test against adults of Asian citrus psyllid (Diaphorina citri) and found to induce 50% reduction in the survival rate of D. citri adults within 5 days of exposure. The ability of the three fungal strains to endophytically colonize Citrus limon, the impact on plant growth and the effects of systemic colonization on 3 successive generations of D. citri feeding on colonized plants was evaluated. Citrus seedlings at 4 months post-planting were inoculated with each of the fungal strains via foliar spraying. Both strains of B. bassiana successfully colonized the seedlings. One of the B. bassiana strains (BB Fafu-13) was sustained up to 12 weeks in the colonized seedlings, whereas the other B. bassiana strain (BB Fafu-16) was only recovered up to 8 weeks post-inoculation. Isaria fumosorosea (IF Fafu-1) failed to colonize the plant. Both strains of B. bassiana induced significant improvement in plant height and flush production in endophytically colonized seedlings. In addition, endophytic B. bassiana caused 10-15% D. citri adult mortality within 7 days of exposure. Female D. citri feeding on B. bassiana challenged plants laid fewer eggs as compared to those feeding on endophyte-free seedlings, while reduction in adult emergence was recorded on B. bassiana treated plants. With this study, we present the first evidence of B. bassiana artificial establishment as fungal endophyte in citrus plants and its negative effects on D. citri.

Role of Saponins in Plant Defense Against Specialist Herbivores.

The diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae) is a very destructive crucifer-specialized pest that has resulted in significant crop losses worldwide. DBM is well attracted to glucosinolates (which act as fingerprints and essential for herbivores in host plant recognition) containing crucifers such as wintercress, Barbarea vulgaris (Brassicaceae) despite poor larval survival on it due to high-to-low concentration of saponins and generally to other plants in the genus Barbarea. B. vulgaris build up resistance against DBM and other herbivorous insects using glucosinulates which are used in plant defense. Aside glucosinolates, Barbarea genus also contains triterpenoid saponins, which are toxic to insects and act as feeding deterrents for plant specialist herbivores (such as DBM). Previous studies have found interesting relationship between the host plant and secondary metabolite contents, which indicate that attraction or resistance to specialist herbivore DBM, is due to higher concentrations of glucosinolates and saponins in younger leaves in contrast to the older leaves of Barbarea genus. As a response to this phenomenon, herbivores as DBM has developed a strategy of defense against these plant biochemicals. Because there is a lack of full knowledge in understanding bioactive molecules (such as saponins) role in plant defense against plant herbivores. Thus, in this review, we discuss the role of secondary plant metabolites in plant defense mechanisms against the specialist herbivores. In the future, trials by plant breeders could aim at transferring these bioactive molecules against herbivore to cash crops.

Molecular characterization of pathogenesis involving the GAS 1 gene from Entomopathogenic fungus Lecanicillium lecanii and its virulence against the insect host Diaphorina citri.

The entmopathogenic fungus Lecaniicillium lecanii is a naturally available biological control and it is considered to be one of the best mycoinsecticide agents against the destructive insect pest Diaphorina citri Kuwayama. The present study aimed to extract and characterize the toxic insecticidal protein from L. lecanii and to assess the toxicity level against the Asian citrus psyllid the vector of Huanglongbing disease (HLB), also called citrus greening. Extracts of a toxic substance from submerged batch culture examined by sodium dodecyl sulfate-poly-acrylamide (SDS-PAGE), had a molecular weight of 45 kDa. The most abundant toxic metabolite was subjected to HPLC to purify and identified it by mass spectrometry. Subsequently, metabolite toxicity was tested against D. citri at three different concentrations (1%, 2%, and 3%). The results showed that the highest concentration had a significant maximum mortality at 120 h post application. Furthermore, we investigated the expression of the GAS1 gene which was previously identified to have a role in pathogenicity in in vivo studies in adult insect psyllids. Results of this study indicated that expression of the virulence factor gene was present at three concentrations of the fungal suspension post inoculation. This is the first study to provide this novel approach for the characterization of fungal mediated synthesis of a cuticle degrading soluble protein against the insect D. citri. The present results provide strong information on the in vivo expression of the GAS1 gene involved in fungal virulence pertaining to penetration of the insect cuticle, but not to inhibiting the growth of the host.

Synthesis of mycotoxin protein IF8 by the entomopathogenic fungus Isaria fumosorosea and its toxic effect against adult Diaphorina citri.

Entomopathogenic fungi based microbial insecticides are considered as safe alternatives to chemical pesticides, which secretes several bioactive compounds to kill the host insects. In this study, we report a new approach for the synthesis and characterization of insecticide toxic protein IF8 produced by the Isaria fumosorosea 08, and to evaluate the mycotoxin level against the vector of Huanglongbing (HLB) or citrus greening disease, the Asian citrus psyllid, Diaphorina citri. Soluble toxic metabolites extracted from I. fumosorosea 08 through submerged liquid state culture had a molecular weight of 43 kDa when subjected by to sodium dodecyl sulfate-poly-acrylamide (SDS-PAGE) gel electrophoresis. The most abundant of toxic protein IF8 was determined by High-performance liquid chromatography (HPLC) and liquid chromatography electrospray ionization-mass spectroscopy (LC-ESI-MS) for the analysis of its molecular mass weight and purity. Further Matrix-assisted laser desorption ionization-time of flight (MALDI-TOFF) analysis confirmed the presence of toxic metabolites in liquid culture. Subsequently, mycotoxic effect of toxic protein IF8 was tested against D. citri at three different concentrations (1%, 2%, and 3%). The results showed the insecticidal activity of >80% when administered at three different concentrations at 48-120 hour post-application. Additionally, we also investigated the physicochemical properties and stability of IF8 by using computational biological tools. This is the first study to report the characterization of fungal mediated synthesis of the protein IF8 toxic to the insect D. citri. These results suggest the mycotoxin control of D. citri and prevention of HLB transmission by using a natural toxic compound which is eco-friendly and can be potentially used for the integrated management of D. citri.