Chandra Kanta Dash

Effects of different temperature regimes on survival of Diaphorina citri and its endosymbiotic bacterial communities

The Asian citrus psyllid, Diaphorina citri, is a major pest of citrus and vector of citrus greening (huanglongbing) in Asian. In our field-collected psyllid samples, we discovered that Fuzhou (China) and Faisalabad (Pakistan), populations harbored an obligate primary endosymbiont Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.) and a secondary endosymbiont, Wolbachia surface proteins (WSP) which are intracellular endosymbionts residing in the bacteriomes. Responses of these symbionts to different temperatures were examined and their host survival assessed. Diagnostic PCR assays showed that the endosymbionts infection rates were not significantly reduced in both D. citri populations after 24 h exposure to cold or heat treatments. Although quantitative PCR assays showed significant reduction of WSP relative densities at 40°C for 24 h, a substantial decrease occurred as the exposure duration increased beyond 3 days. Under the same temperature regimes, Ca. C. ruddii density was initially less affected during the first exposure day, but rapidly reduced at 3-5 days compared to WSP. However, the mortality of the psyllids increased rapidly as exposure time to heat treatment increased. The responses of the two symbionts to unfavorable temperature regimes highlight the complex host-symbionts interactions between D. citri and its associated endosymbionts.

Investigation and molecular docking studies of Bassianolide from Lecanicillium lecanii against Plutella xylostella (Lepidoptera: Plutellidae)

Entomopathogenic fungi are rich sources of bioactive secondary metabolites that possess insecticidal properties. The present study reported a novel approach for the identification of insecticidal compounds produced by Lecanicillium lecanii 09 and to assess their toxicity against the diamondback moth Plutella xylostella L. The cyclic peptides groups of toxic substances were separated from L. lecanii 09 through submerged liquid state fermentation. The most abundant toxic metabolite, Bassianolide was purified by high-performance liquid chromatography (HPLC) and its molecular weight and purity were determined by Liquid chromatography - mass spectroscopy (LC-MS), Fourier transformed infrared spectroscopy (FT-IR), and H1 nuclear magnetic resonance (NMR) respectively. Subsequently, the toxicity of bassianolide was tested against third instar larvae of P. xylostella at three different concentrations (0.01, 0.1, 0.5 mg/ml). The results showed that higher concentration of 0.5 mg/ml had significant maximum mortality at 120 hour post inoculation. Furthermore, we investigated the ligand-target interaction of secondary metabolite binding with target insect immune receptor proteins and predicted the role of toxicity against insect host. This is the first study to report the infection process and the interaction of fungal mediated cyclicdepsipeptide compound (bassianolide) from L. lecanii 09 against the insect host P. xylostella. This novel approach provides a potential impact on biological control using natural toxic compound which acts as good inhibitor on pest insect and prevents toxicity hazards, pollution as well as ecocidal effects killing several beneficial insects.

Phylogenetic relationship and genetic diversity of citrus psyllid populations from China and Pakistan and their associated Candidatus bacterium

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a severely devastating pest of Rutaceae plants, mainly citrus, across the globe, and causal agent of Huanglongbing (HLB) disease. To find out the genetic relationship and diversity among the populations of ACP and associated Candidatus Liberibacter asiaticus (CLas) from two countries (China and Pakistan), sequence data of three different genes, cytochrome oxidase subunit I (COI), Cu-transporting protein (ATOX1) and 16S rRNA, were used to characterize all populations. In the present study, MEGA-7 and statistical parsimony software (TCS-1.2) were used to depict the phylogenetic relationship among all populations under both genes, whereas diversity was calculated by DnaSP v5. All analyses were done for country wise and overall relationship among all populations. For ACP populations, both genes presented a significant strong intermingled relationship among all populations and put all population into a single haplotype (Dcit-2), which proved similarity between Chinese and Pakistani populations. Moreover, for CLas strains, 16S gene also presented strong relationship for all sampled populations. All three genes of ACP and CLas populations elucidated more than 95% resemblance to each other. On the other hand, a significant genetic variation was observed by three genes for overall populations, although, country wise variation was non-significant between all collected populations. ATOX1 gene presented higher diversity through Fus Fs test (π = 0.01081, p < 0.003) whereas COI gene gave less diversity under Fus Fs and Tajimas D test (π = 0.00512, p < 0.000 and 0.05, respectively). Similarly, nucleotide mismatch distribution also had shown enough genetic variation among all ACP populations, under both genes. Our sequence data for both genes proved the invasion of the Chinese ACP population (Dcit-2) into Pakistan, through all phylogenetic relationship, which proved a similar genetic makeup among all ACP populations from both countries. Therefore, these results can be helpful to utilize any novel designed control measure equally for both countries.

Isolation and characterization of Metarhizium anisopliae TK29 and its mycoinsecticide effects against subterranean termite Coptotermes formosanus

The entomopathogenic fungus Metarhizium anisopliae is widely used as biocontrol agent against many insect pests. In the present study, the potential isolate of M. anisopliae TK29 was isolated from the agricultural soils in Thekkady, India. The taxonomic identity of the isolate was confirmed based on its morphology and 18S rDNA gene sequence homology. Phylogenetic analysis confirmed that the isolated strains were related to the same species. A potential isolate (TK29) was optimized for mass cultivation and conidial spore production was enhanced using three different raw substrates (Rice, Maize, black gram) by solid-state fermentation. The results showed higher conidial spore yield from rice (2.6 ± 0.32%) compared to black gram (2.1 ± 0.28%) and maize (1.9 ± 0.23%) substrates. Dry green conidia were applied against Formosan subterranean termite, Coptotermes formosanus at three different concentrations (1 × 106, 1 × 107, and 1 × 108 conidia/ml-1). The highest mortality rate was obtained from 1 × 108 conidia/ml-1 at 120 h post-treatment. Our study indicated that M. anisopliae TK29 had desirable attributes for the development of a mycoinsecticide against C. formosanus.

Fungal Endophytes: Beyond Herbivore Management

The incorporation of entomopathogenic fungi as biocontrol agents into Integrated Pest Management (IPM) programs without doubt, has been highly effective. The ability of these fungal pathogens such as Beauveria bassiana and Metarhizium anisopliae to exist as endophytes in plants and protect their colonized host plants against the primary herbivore pests has widely been reported. Aside this sole role of pest management that has been traditionally ascribed to fungal endophytes, recent findings provided evidence of other possible functions as plant yield promoter, soil nutrient distributor, abiotic stress and drought tolerance enhancer in plants. However, reports on these additional important effects of fungal endophytes on the colonized plants remain scanty. In this review, we discussed the various beneficial effects of endophytic fungi on the host plants and their primary herbivore pests; as well as some negative effects that are relatively unknown. We also highlighted the prospects of our findings in further increasing the acceptance of fungal endophytes as an integral part of pest management programs for optimized crop production.

Genetic diversity of the families Aeshnidae, Gomphidae and Libellulidae through COI gene from South China

Adult dragonflies (Anisoptera) were collected from different localities of South China covering eight provinces. Representative sequences were sixty-one, including 16 species, 11 genera and three families (Aeshnidae, Gomphidae and Libellulidae), under cytochrome oxidase subunit I (COI) gene. After alignment of sequences by BioEdit v6, genetic interaction and divergence were computed by MEGA 7 whereas all the indices of genetic diversity were calculated by DnaSP v5 software. Phylogenetic trees were constructed through Neighbor-Joining method under Jukes-Cantor model, and all species of respective families were assembled with each other into individual groups. Maximum divergence was observed by Trithemis genus (18.69%), followed by Orthetrum genus (18.16%), whereas a minimum value of divergence was noted for Pantala genus (0.31%). On the other hand, maximum genetic diversity was recorded for Orthetrum genus up to 142 mutations, followed by Trithemis genus (126 mutations), while the minimum value (two mutations) was observed for Pantala genus. Genetic diversity for overall and Libellulidae family sequences was much higher, up to 404 mutations and 344 mutations, respectively. Current results suggest a high diversity of odonates in the South China region and results are valuable in gaining a total obligation of the diversity of Asian odonates and conservation measures of this insect group.

Effect of vineyard soil variability on chlorophyll fluorescence, yield and quality of table grape as influenced by soil moisture, grown under double cropping system in protected condition

Environmental factors greatly influence grape quality. Among them, the effect of within-vineyard variability of soil in relation to soil moisture on table grape under protected condition has rarely been studied. In this present research, we investigated the influence of soil variability, in relation with soil moisture on chlorophyll fluorescence, yield and quality attributes of the “Summer Black” (Vitis vinifera L. × V. labruscana L.) table grape, popularly grown under double cropping system in protected covering in the southern part of China. The vineyard was divided vertically into three sites (lower, middle and upper, 192, 202 and 212 m above sea level, respectively) and data on soil moisture and other yield and quality parameters were recorded. Among the three vineyard sites, middle site resulted in higher yield compared to the upper and lower sites during winter and summer cropping cycles. However, compared to regular summer cycle, winter cycle provided grapevines with higher quality attributes. Polyphasic OJIP fluorescence transient exhibited a considerable increase in fluorescence intensity at J, I and P phase in the upper and middle sites compared to the lower site due to variation in soil moisture in both seasons. Values of fluorescence parameters including minimal fluorescence, relative variable fluorescence at phase J and I, the maximal quantum yield of photosystem II were also influenced by soil moisture in different sites. Different sites also exhibited a significant difference in total phenolics, flavonoid, antioxidant activity and individual anthocyanin which was influenced by available soil moisture. The present study shows that chlorophyll fluorescence OJIP transient can be used as a sensitive indicator to determine the moisture stress in grape grown in a varied soil. Double cropping proved to be a powerful technique to improve the fruit quality. This result may be useful for the table grape growers to better utilize the vineyard soil variability with water management to get higher yield and quality table grape under protected condition.

Prospects of endophytic fungal entomopathogens as biocontrol and plant growth promoting agents: An insight on how artificial inoculation methods affect endophytic colonization of host plants

Entomopathogenic fungi (EPF) can be established as endophytes in the host plants to offer a long-term preventive measure for pests and diseases. This practice serves as a better alternative to the common practice of periodic direct application of EPF on plants or the target pests as a short-term defense strategy against pests and diseases. These fungal endophytes, aside from their role in pests and diseases prevention, also act as plant growth promoters. Several fungal endophytes have been associated with improvement in plant height, dry and wet weight and other growth parameters. However, many limiting factors have been identified as mitigating the successful colonization of the host plants by EPF. The inoculation methods used have been identified as one, but sadly, this has received little or less attention. Some previous studies carried out comparison between various artificial inoculation methods; foliar application, seedling dipping, soil drenching, seed inoculation, direct injection and others. In separate studies, some authors had suggested different application methods that are best suitable for certain fungal entomopathogens. For instance, leaf inoculation with conidial suspensions was suggested to be the best inoculation method for Beauveria bassiana in sorghum, stem injection was suggested as the most suitable for coffee, while, root dipping method proved the most successful for B. bassiana colonization of tomato plants for the management of Helicoverpa armigera. Here, we discussed entomopathogenic fungal endophytes as bio-control agents, plant growth promoters and highlighted the effect of various artificial inoculation methods on their endophytic colonization of the host plants.

Endophytic entomopathogenic fungi enhance the growth of Phaseolus vulgaris L. (Fabaceae) and negatively affect the development and reproduction of Tetranychus urticae Koch (Acari: Tetranychidae)

Entomopathogenic fungi (EPF) have primarily been applied as an inundative approach to manage pests. However, in recent decade multifunctional role of EPF have been documented which provide multiple benefits to host plants when colonized as an endophyte. In this study five fungal isolates from the genus Beauveria (three), Isaria (one) and Lecanicillium (one) were evaluated for their ability to colonize common bean, Phaseolus vulgaris and to assess their effects in planta on plant growth promotion and possible negative effects on the two-spotted spider mites, Tetranychus urticae. All the tested isolates in this study were able to endophytically colonize root, stem and even leaves of inoculated plants examined at 7 and 14 days post inoculation, indicating the systemic colonization of EPF. Colonized plants showed increased plant heights, fresh shoot and root weights compared to plants without inoculation. Survivorship of T. urticae significantly differed among the treatments with higher survival probability in control plants. Significant reduction in larval development, adult longevity and female fecundity of spider mites were observed when fed on treated plants compared to control plants. The negative effects were found to be carried over the second generation fed on fresh plants. Overall, our results show (i) the positive effects of fungal endophytes on plant growth, (ii) reduction in population growth rate and (iii) negative effects of endophytes on growth and reproduction of spider mites in successive generations. The study presents reports on the endophytic management of plant-feeding mites and highlights the possibility of utilizing entomopathogenic fungal endophytes in the integrated pest management program.

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.