Microbial and viral chitinases: Attractive biopesticides for integrated pest management.

The virulence of eight isolates of Metarhizium anisopliae and four isolates of Beauveria bassiana (Ascomycota: Hypocreales) to adult Cylas puncticollis (Coleoptera: Curculionidae) was tested in the laboratory. Insects were sprayed with a standard concentration of 1.0 x 10 7 conidia mL -1 using Burgerjon's spray tower. All the isolates tested were pathogenic to C. puncticollis. Mortality varied between 77.5% and 84.2% with isolates of B. bassiana and between 62.5% and 89.2% with isolates of M. anisopliae, 26 days post-treatment. The lethal time to 50% mortality for the 12 isolates varied between 9.7 and 18.5 days. Four isolates, M. anisopliae International Centre of Insect Physiology and Ecology (ICIPE) 18 and ICIPE 62 and B. bassiana ICIPE 275 and ICIPE 114 were selected for dose-response mortality [lethal concentration to 50% mortality (LC 50 )] bioassays. Five concentrations (1.0 x 10 6 , 3.0 x 10 6 , 1.0 x 10 7 , 3.0 × 10 7 and 1.0 x 10 8 conidia mL -1 ) of both fungal species were used. B. bassiana ICIPE 275 was the most active isolate with LC 50 value of 0.7 x 10 6 conidia mL -1 . The effect of fungal infection on feeding, fecundity and egg viability of C. puncticollis adult females was also investigated under laboratory conditions. M. anisopliae isolate ICIPE 18 and B. bassiana isolate ICIPE 114 were tested for feeding experiment using six concentrations (0, 1.0 x 10 6 , 3.0 x 10 6 , 1.0 x 10 7 , 3.0 x 10 7 and 1.0 x 10 8 conidia mL -1 ). For reproduction potential (fecundity and egg viability) bioassays, five concentrations (0, 1.0 x 10 6 , 3.0 x 10 6 , 1.0 x 10 7 and 3.0 x 10 7 conidia mL -1 ) of M. anisopliae isolate ICIPE 18 were used. Adult sweet potato weevils (SPWs) treated with M. anisopliae at the concentrations of 3.0 x 10 7 and 1.0 x 10 8 conidia mL -1 consumed significantly less food than weevils in the control and B. bassiana treatments at all the concentrations, except at the higher concentration of 1.0 x 10 8 conidia mL -1 , 14 days post-treatment. Female weevils in the control treatments laid more eggs than fungus-treated females. Percentage egg viability differences between controls and fungus treatments were significant at all the concentrations tested, 10 days post-treatment. These results show that B. bassiana and M. anisopliae are pathogenic to SPWs and infection can reduce feeding, fecundity and egg viability.

Virulence of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to sweet patato weevil Cylas puncticollis and effects on fecundity and egg viability

The emergence of insecticide resistant insect pests is of significant concern worldwide. The whitefly, Bemisia tabaci, is an important agricultural pest and has shown incredible resilience developing resistance to a number of chemical pesticides. Entomopathogenic fungi such as Isaria fumosorosea offer an attractive alternative to chemical pesticides for insect control, and this fungus has been shown to be an effective pathogen of B. tabaci. Little is known concerning the potential for the development of resistance to I. fumosorosea by B. tabaci. Five generations of successive survivors of B. tabaci infected by I. fumosorosea were assayed with I. fumosorosea. No significant differences in susceptibility to I. fumosorosea, number of ovarioles, or ovipostioning were seen between any of the generations tested. Effects of I. fumosorosea and cell-free ethyl acetate fractions derived from the fungus on the B. tabaci fat body, ovary, and vitellogenin were also investigated. These data revealed significant deformation and degradation of ovary tissues and associated vitellogenin by the fungal mycelium as well as by cell-free ethyl acetate fungal extracts. These data indicate the lack of the emergence of resistance to I. fumosorosea under the conditions tested and demonstrate invasion of the insect reproductive tissues during fungal infection.

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Lack of resistance development in Bemisia tabaci to Isaria fumosorosea after multiple generations of selection.

Dual biocontrol of both insects and plant pathogens has been reported for certain fungal entomopathogens, including Beauveria bassiana and Lecanicillum spp. In this study, we demonstrate, for the first time, the dual biocontrol potential of two fungal isolates identified by morphological and phylogenetic analyses as Isaria javanica. Both these isolates caused mortality in the greater wax moth, and hence can be considered entomopathogens. Spores of the isolates were also pathogenic to nymphs of the green peach aphid (Myzus persicae), with an LC50 value of 107 spores/mL 4 days after inoculation and an LT50 of 4.2 days with a dose of 108 spores/mL. In vitro antifungal assays also demonstrated a strong inhibitory effect on the growth of two fungi that are pathogenic to peppers, Colletotrichum gloeosporioides and Phytophthora capsici. These results indicate that I. javanica isolates could be used as novel biocontrol agents for the simultaneous control of aphids and fungal diseases, such as anthracnose and Phytophthora blight, in an integrated pest management framework for red pepper.

Dual Biocontrol Potential of the Entomopathogenic Fungus, Isaria javanica, for Both Aphids and Plant Fungal Pathogens

Serratia marcescens is a bacterium with the ability to colonize several niches, including some eukaryotic hosts. S. marcescens have been recently found in the gut of hematophagous insects that act as parasite vectors, such as Anopheles, Rhodnius, and Triatoma. While some S. marcescens strains have been reported as symbiotic or pathogenic to other insects, the role of S. marcescens populations from the gut microbiota of Rhodnius prolixus, a vector of Chagas' disease, remains unknown. Bacterial colonies from R. prolixus gut were isolated on BHI agar. After BOX-PCR fingerprinting, the genomic sequences of two isolates RPA1 and RPH1 were compared to others S. marcescens from the NCBI database in other to estimate their evolutionary divergence. The in vitro trypanolytic activity of these two bacterial isolates against Trypanosoma cruzi (DM28c clone and Y strain) was assessed by microscopy. In addition, the gene expression of type VI secretion system (T6SS) was detected in vivo by RT-PCR. Comparative genomics of RPA1 and RPH1 revealed, besides plasmid presence and genomic islands, genes related to motility, attachment, and quorum sensing in both genomes while genes for urea hydrolysis and type II secretion system (T2SS) were found only in the RPA1 genome. The in vitro trypanolytic activity of both S. marcescens strains was stronger in their stationary phases of growth than in their exponential ones, with 65-70 and 85-90% of epimastigotes (Dm28c clone and Y strain, respectively) being lysed after incubation with RPA1 or RPH1 in stationary phase. Although T6SS transcripts were detected in guts up to 40 days after feeding (DAF), R. prolixus morbidity or mortality did not appear to be affected. In this report, we made available two trypanolytic S. marcescens strains from R. prolixus gut to the scientific community together with their genomic sequences. Here, we describe their genomic features with the purpose of bringing new insights into the S. marcescens adaptations for colonization of the specific niche of triatomine guts. This study provides the basis for a better understanding of the role of S. marcescens in the microbiota of R. prolixus gut as a potential antagonist of T. cruzi in this complex system.

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In vitro Trypanocidal Activity, Genomic Analysis of Isolates, and in vivo Transcription of Type VI Secretion System of Serratia marcescens Belonging to the Microbiota of Rhodnius prolixus Digestive Tract.

The filamentous fungus, Isaria fumosorosea, is a promising insect biological control agent. Chitinases have been implicated in targeting insect cuticle structures, with biotechnological potential in insect and fungal control. The I. fumosorosea chitinase gene, Ifchit1, was isolated and determined to encode a polypeptide of 423 amino acids (46 kDa, pI = 6.53), present as a single copy in the I. fumosorosea genome. A split marker transformation system was developed and used to construct an Ifchit1 gene knockout. The ΔIfchit1 strain displayed minor alterations in mycelial growth on diverse media at 26 °C compared to the wild type and complemented (ΔIfchit1::Ifchit1) strains; however, colony morphology was affected, and the mutant strain had a temperature sensitive phenotype (32 °C). Although sporulation was delayed for the mutant, overall conidial production was almost twice than that of wild type. Biochemical assays indicated decreased chitinase activity during growth in Czapek-Dox liquid media for the ΔIfchit1 strain. Insect bioassays using diamondback moth, Plutella xylostella, larvae revealed decreased infectivity, i.e., increased LC
 50
 (threefold to fourfold) and a significantly delayed time to death, LT
 50
 from 3 to 6 days, for the ΔIfchit1 strain compared to the wild type and complemented strains. These data indicate an important role for the Ifchit1 chitinase as a virulence factor in I. fumosorosea.

The Ifchit1 chitinase gene acts as a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea.

Background The genomes of broad host range insect pathogenic fungi, including Cordyceps fumosorosea, encode for a suite of secreted proteases implicated in targeting, penetration, and degradation of the host exoskeleton or cuticle. These cuticle-degrading proteases act as critical virulence factors, but their functions within the biological context, particularly in relation to host specificity, remain poorly characterized. Result A C. fumosorosea protease gene, Cfcdp1, was identified and a targeted gene-knockout strain constructed. Minor growth defects were observed for the ΔCfcdp1 strain when compared to the wild-type parent and complemented (ΔCfcdp1::Cfcdp1) strains, with delayed and decreased sporulation noted for the mutant. Decreased subtilisin-like protease activity was seen for the ΔCfcdp1 strain, although total secreted protease activity was similar between the mutant and wild-type strains. Insect bioassays using whitefly, Bemisia tabaci, and cabbageworm, Pieris rapae, showed decreased infectivity, i.e. 2.4-3.4-fold increase in lethal dose (LC50 ) and an increased time to death (LT50 ), for the ΔCfcdp1 strain. In contrast, insect bioassays using the diamondback moth, Plutella xylostella, or the brown planthopper, Nilaparvata lugens, showed increased infectivity, i.e. a 3-5-fold decrease in LC50 , and a decreased LT50 . Differential effects were also seen on the fecundity of B. tabaci infected by the different fungal strains. Conclusion These data reveal host-dependent effects of a protease implicated in cuticle degradation on C. fumosorosea virulence. The implications of these findings in suggesting context-dependent requirements of cuticle-degrading enzymes and their potentially differential roles in mediating virulence towards different hosts are discussed. © 2019 Society of Chemical Industry.

Yongfen Hao

Papers

The Ifchit1 chitinase gene acts as a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea.

Host-dependent contributions of the Cfcdp1 protease gene to virulence in the entomopathogenic fungus Cordyceps fumosorosea.