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Biocontrol agents can control pathogens by reenforcing systemic plant resistance through systemic acquired resistance (SAR) or induced systemic resistance (ISR). Trichoderma spp. can activate the plant immune system through ISR, priming molecular mechanisms of defense against pathogens. Entomopathogenic fungi (EPF) can infect a wide range of arthropod pests and play an important role in reducing pests’ population. Here, we investigated the mechanisms by which EPF control plant diseases. We tested two well studied EPF, Metarhizium brunneum isolate Mb7 and Beauveria bassiana as the commercial product Velifer, for their ability to induce systemic immunity and disease resistance against several fungal and bacterial phytopathogens, and their ability to promote plant growth. We compared the activity of these EPF to an established biocontrol agent, Trichoderma harzianum T39, a known inducer of systemic plant immunity and broad disease resistance. The three fungal agents were effective against several fungal and bacterial plant pathogens and arthropod pests. Our results indicate that EPF induce systemic plant immunity and disease resistance by activating the plant host defense machinery, as evidenced by increases in reactive oxygen species production and defense gene expression, and that EPF promote plant growth. EPF should be considered as control means for Tuta absoluta. We demonstrate that, with some exceptions, biocontrol in tomato can be equally potent by the tested EPF and T. harzianum T39, against both insect pests and plant pathogens. Taken together, our findings suggest that EPF may find use in broad-spectrum pest and disease management and as plant growth promoting agents.

Biocontrol agents can control pathogens by reenforcing systemic plant resistance through systemic acquired resistance (SAR) or induced systemic resistance (ISR).

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.

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.

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.The parasitism rate and host-feeding rate ofTamarixia radiata(Hymenoptera: Eulophidae), an ectoparasitoid ofDiaphorina 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 ofT. radiatawas evaluated by linking the age-stage predation rate with the two-sex life table. The net host-feeding rate (C0) byT. radiatawas 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 ofT. radiatawere 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.

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.

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.

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&ndash, 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.

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.

In the present study, commercially important crop Carica papaya latex extract was used as potential bioreductant to fabricate high-stable silver nanoparticles (CPAgNPs). The reduction of Ag + in Ag 0 nanostructures was initially observed with a color change from light to dark brown and further confirmed through SPR spectra at 410 nm in UV–vis spectroscopy. Fourier transform infrared spectroscopy (FTIR) analysis reveals that protein moieties present in the latex extract act as reducing/capping agent for nanoparticle synthesis. Interestingly, X-ray diffraction (XRD) study confirms that synthesized CPAgNPs were face centered cubic crystalline (fcc) in structure. High resolution transmission electron microscope (HRTEM) micrographs display polydispersed spherical CPAgNPs with an average size of 12 ± 6 nm. The CPAgNPs display tremendous antibacterial activity against human pathogens such as Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae and Proteus mirabilis . On the other hand, in vitro anticancer studies were performed to assess the chemotherapeutic potential of formulating NPs against human breast carcinoma cell line (MCF-7). It was observed that fabricated CPAgNPs show excellent anticancer effect in a dose-dependent manner with the IC 50 value 19.88 μg/ml. These results clearly impelled that biogenic CPAgNPs can be used as promising nano-drug formulation to combat against bacterial infections and also for breast cancer chemotherapy

Fungal virulence has been mostly associated with cuticle-degrading enzymes, which form the first formidable barrier to pathogens and pass through certain discrete stages before breaching the insect cuticle. The present study was conducted to extract and purify the extracellular protease enzyme from three isolates from Metarhizium anisopliae. The molecular weight of protease enzyme from each isolate was identified using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and found to be 35-40kDa. The partially purified enzymes were tested to identify its toxic effects against the developmental stages of IVth instar larvae of Galleria mellonella and the mortality of larvae among the three isolates was observed. The Tk6 isolate showed an ascending effect after 48h of exposure, with highest mortality at 120h post inoculation. It also showed more virulence against the model insect compared to other strains. Tk6 isolate's active protein band was analyzed by MALDI-TOF and docking study was carried out to find the interaction between the fungal and insect proteins.