Bacterial biota associated with the invasive insect pest Tuta absoluta (Meyrick).

Tuta absoluta (the tomato pinworm) is an invasive insect pest with a highly damaging effect on tomatoes causing between 80 and 100% yield losses if left uncontrolled. Resistance to chemical pesticides have been reported in some T. absoluta populations. Insect microbiome plays an important role in the behavior, physiology, and survivability of their host. In a bid to explore and develop an alternative control method, the associated microbiome of this insect was studied. In this study, we unraveled the bacterial biota of T. absoluta larvae and adults by sequencing and analyzing the 16S rRNA V3-V4 gene regions using Illumina NovaSeq PE250. Out of 2,092,015 amplicon sequence variants (ASVs) recovered from 30 samples (15 larvae and 15 adults), 1,268,810 and 823,205 ASVs were obtained from the larvae and adults, respectively. A total of 433 bacterial genera were shared between the adults and larval samples while 264 and 139 genera were unique to the larvae and adults, respectively. Amplicon metagenomic analyses of the sequences showed the dominance of the phylum Proteobacteria in the adult samples while Firmicutes and Proteobacteria dominated in the larval samples. Linear discriminant analysis effect size (LEfSe) comparison revealed the genera Pseudomonas, Delftia and Ralstonia to be differentially enriched in the adult samples while Enterococcus, Enterobacter, Lactococcus, Klebsiella and Wiessella were differentially abundant in the larvae. The diversity indices showed that the bacterial communities were not different between the insect samples collected from different geographical regions. However, the bacterial communities significantly differed based on the sample type between larvae and adults. A co-occurrence network of significantly correlated taxa revealed a strong interaction between the microbial communities. The functional analysis of the microbiome using FAPROTAX showed that denitrification, arsenite oxidation, methylotrophy and methanotrophy as the active functional groups of the adult and larvae microbiomes. Our results have revealed the core taxonomic, functional, and interacting microbiota of T. absoluta and these indicate that the larvae and adults harbor a similar but transitory set of bacteria. The results provide a novel insight and a basis for exploring microbiome-based biocontrol strategy for this invasive insect pest as well as the ecological significance of some of the identified microbiota is discussed.

Heterologous array analysis in Heterobasidion: hybridisation of cDNA arrays with probe from mycelium of S, P or F-types.

Because of the close relatedness between three species of Heterobasidion annosum (P-type), Heterobasidion parviporum (S-type) and Heterobasidion abietinum (F-type), we investigated the possible use of arrays from one species for studies of gene expression in the other. Clones containing partial cDNAs from 94 identifiable genes expressed during spore germination and differentiation in H. parviporum were printed manually in six replications on nylon membranes. The membrane was hybridized with chemifluorescent labelled cDNA from actively growing mycelia of H. parviporum, H. annosum or H. abietinum, cultivated on a non-selective substrate. Product-moment correlation coefficient varied between 0.81 and 0.49. Due to the level of correlation, in the gene expression among the intersterility groups, we concluded that the cDNA array of one can be used to study gene expression in the others.

Effects of carbohydrates, ethanol and selected cell wall phenolics on in vitro growth of necrotrophic fungi--Heterobasidion annosum and Fusarium avenaceum.

In this study, a comparative assessment was undertaken of nutritional factors that are likely to influence in vivo growth of the S and P-types of necrotrophic root rot fungus Heterobasidion annosum and of the damping off pathogen Fusarium avenaceum respectively. The results indicate that glucose and fructose but not cellobiose or sucrose supported growth of these fungi when used as sole carbon source. Particularly for P-types and F. avenaceum, however, the utilization of cellobiose and sucrose was considerably enhanced by several fold in the presence of ethanol. In addition, key plant cell wall phenolics (ferulic acid, caffeic acid, p-coumaric acid) at 0.025% inhibited growth of the test fungi. But this toxicity was reversed in the presence of exogenously added cell wall sugars (glucose, fructose, sucrose, cellobiose) except for the P-types where sucrose addition was apparently slightly effective. Similarly, ethanol whether alone or in combination with the phenolics was poorly utilized except for F. avenaceum. The importance of these results in understanding factors influencing growth and development during pathogenesis of the necrotrophs on their conifer hosts is discussed.

Inhibition of cellulose saccharification and glycolignin-attacking enzymes of five lignocellulose-degrading fungi by ferulic acid.

At 5 g/l, ferulic acid, a plant cell-wall phenolic, severely repressed growth of the lignocellulose-degrading fungi Trichoderma harzianum, Chaetomium cellulolyticum, Phanaerochaete chrysosporium, Trametes versicolor and Pleurotus sajor-caju. At 0.5 g/l, howerver, it slightly stimulated growth of the latter two organisms. Two classes of extracellular enzymes involved in cellulose and glycolignin breakdown were assayed: cellulases; and phenol oxidases as laccases. All of the strains depolymerized cellulose but two (T. versicolor and P. sajor-caju) also secreted laccases. Laccase-secreting fungal species had normal levels of cellulose saccharification except in the presence of 5 g ferulic acid/l, whereas saccharification by the other strains was suppressed at all concentrations of the phenolic tested.

The effects of co-fungal cultures and supplementation with carbohydrate adjuncts on lignin biodegradation and substrate digestibility.

The ability of three fungal strains (Pleurotus sajor-caju, Phanerochaete chrysosporium, Trametes versicolor) to decrease the lignin content and to enhance in vitro rumen digestibility of lignified spruce sawdust was assessed. In monoculture solid substrate fermentation (SSF) studies, a considerable length of time (6 weeks) elapsed before 4 to 14% lignin was degraded. In contrast, paired or multiple cultures of these fungi caused an 8 to 16% loss of native lignin within three weeks of incubation. There were also synergistic effects on total polysaccharide/hemicellulose degraded by mixed cultures. A similar observation was made for in vitro digestibility of fungal fermented samples: Total solubles (carbohydrate products) which accumulated in cultures were significantly higher in mixed cultures than in respective monocultures. In contrast, mixtures of cell free enzyme extracts of these fungi did not cause any marked reduction in lignin or cellulose content. Supplementation of wood sawdust with carbohydrate adjuncts prior to fungal treatment also led to substantial reduction in lignin content and increased substrate digestibility.

Infection and disintegration of vascular tissues of non-suberized roots of spruce byHeterobasidion annosum and use of antibodies for characterizing infection.

Vascular disintegration mainly of medulla rays of spruce roots is of major significance in root rot disease of spruce caused byH. annosum. Using seedling roots as an experimental model, the possible routes and initial host reactions preceding invasion of vascular tissues was investigated. Transmission electron microscopy showed that penetration through the endodermis was an obvious route but not without host resistance. Using antibodies againstH. annosum hyphal materials, some labelling of vascular tissues remote from sites of fungal colonization suggest the release of fungal secretory products partly active in tissue disintegration. Similarly, intense labelling was also observed in severely colonized host tissues at late stages of infection. Strong labelling recorded at 3 d p.i. mainly on fungal hyphae and scant gold particles on invaded host tissues could imply that induction of host antifungal metabolites may have been a late event. A correlation was found between total antigenic material in root homogenates measured by ELISA, density of tissue labelling by immunocytochemistry and severity of disease symptoms. The importance of this in relation to diagnosis of biotic root rot diseases in the field is discussed.