Production and characterization of Trichoderma asperellum chitinases and their use in synergy with Bacillus thuringiensis for lepidopteran control.

BACKGROUND: Despite their known negative effects on ecosystems and human health, synthetic pesticides are still largely used to control crop insect pests. Currently, the biopesticide market for insect biocontrol mainly relies on the entomopathogenic bacterium Bacillus thuringiensis (Bt). New biocontrol tools for crop protection might derive from fungi, in particular from Trichoderma spp., which are known producers of chitinases and other bioactive compounds able to negatively affect insect survival. RESULTS: In this study, we first developed an environmentally sustainable production process for obtaining chitinases from Trichoderma asperellum ICC012. Then, we investigated the biological effects of this chitinase preparation - alone or in combination with a Bt-based product - when orally administered to two lepidopteran species. Our results demonstrate that T. asperellum efficiently produces a multi-enzymatic cocktail able to alter the chitin microfibril network of the insect peritrophic matrix, resulting in delayed development and larval death. The co-administration of T. asperellum chitinases and sublethal concentrations of Bt toxins increased larval mortality. This synergistic effect was likely due to the higher amount of Bt toxins that passed the damaged peritrophic matrix and reached the target receptors on the midgut cells of chitinase-treated insects. CONCLUSION: Our findings may contribute to the development of an integrated pest management technology based on fungal chitinases that increase the efficacy of Bt-based products, mitigating the risk of Bt-resistance development. © 2024 Society of Chemical Industry.

Roles of the MYB94/FUSED LEAVES1 (ZmFDL1) and GLOSSY2 (ZmGL2) genes in cuticle biosynthesis and potential impacts on Fusarium verticillioides growth on maize silks.

Maize silks, the stigmatic portions of the female flowers, have an important role in reproductive development. Silks also provide entry points for pathogens into host tissues since fungal hyphae move along the surface of the silks to reach the site of infection, i.e., the developing kernel. The outer extracellular surface of the silk is covered by a protective hydrophobic cuticle, comprised of a complex array of long-chain hydrocarbons and small amounts of very long chain fatty acids and fatty alcohols. This work illustrates that two previously characterized cuticle-related genes separately exert roles on maize silk cuticle deposition and function. ZmMYB94/FUSED LEAVES 1 (ZmFDL1) MYB transcription factor is a key regulator of cuticle deposition in maize seedlings. The ZmGLOSSY2 (ZmGL2) gene, a putative member of the BAHD superfamily of acyltransferases with close sequence similarity to the Arabidopsis AtCER2 gene, is involved in the elongation of the fatty acid chains that serve as precursors of the waxes on young leaves. In silks, lack of ZmFDL1 action generates a decrease in the accumulation of a wide number of compounds, including alkanes and alkenes of 20 carbons or greater and affects the expression of cuticle-related genes. These results suggest that ZmFDL1 retains a regulatory role in silks, which might be exerted across the entire wax biosynthesis pathway. Separately, a comparison between gl2-ref and wild-type silks reveals differences in the abundance of specific cuticular wax constituents, particularly those of longer unsaturated carbon chain lengths. The inferred role of ZmGL2 is to control the chain lengths of unsaturated hydrocarbons. The treatment of maize silks with Fusarium verticillioides conidia suspension results in altered transcript levels of ZmFDL1 and ZmGL2 genes. In addition, an increase in fungal growth was observed on gl2-ref mutant silks 72 hours after Fusarium infection. These findings suggest that the silk cuticle plays an active role in the response to F. verticillioides infection.

Conserved secreted effectors contribute to endophytic growth and multihost plant compatibility in a vascular wilt fungus.

Fungal interactions with plant roots, either beneficial or detrimental, have a crucial impact on agriculture and ecosystems. The cosmopolitan plant pathogen Fusarium oxysporum (Fo) provokes vascular wilts in more than a hundred different crops. Isolates of this fungus exhibit host-specific pathogenicity, which is conferred by lineage-specific Secreted In Xylem (SIX) effectors encoded on accessory genomic regions. However, such isolates also can colonize the roots of other plants asymptomatically as endophytes or even protect them against pathogenic strains. The molecular determinants of endophytic multihost compatibility are largely unknown. Here, we characterized a set of Fo candidate effectors from tomato (Solanum lycopersicum) root apoplastic fluid; these early root colonization (ERC) effectors are secreted during early biotrophic growth on main and alternative plant hosts. In contrast to SIX effectors, ERCs have homologs across the entire Fo species complex as well as in other plant-interacting fungi, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence and rapid activation of plant immune responses, while ERC deletion in a nonpathogenic isolate led to impaired root colonization and biocontrol ability. Strikingly, some ERCs contribute to Fo infection on the nonvascular land plant Marchantia polymorpha, revealing an evolutionarily conserved mechanism for multihost colonization by root infecting fungi.

Bacterial Communities in the Embryo of Maize Landraces: Relation with Susceptibility to Fusarium Ear Rot.

Locally adapted maize accessions (landraces) represent an untapped resource of nutritional and resistance traits for breeding, including the shaping of distinct microbiota. Our study focused on five different maize landraces and a reference commercial hybrid, showing different susceptibility to fusarium ear rot, and whether this trait could be related to particular compositions of the bacterial microbiota in the embryo, using different approaches. Our cultivation-independent approach utilized the metabarcoding of a portion of the 16S rRNA gene to study bacterial populations in these samples. Multivariate statistical analyses indicated that the microbiota of the embryos of the accessions grouped in two different clusters: one comprising three landraces and the hybrid, one including the remaining two landraces, which showed a lower susceptibility to fusarium ear rot in field. The main discriminant between these clusters was the frequency of Firmicutes, higher in the second cluster, and this abundance was confirmed by quantification through digital PCR. The cultivation-dependent approach allowed the isolation of 70 bacterial strains, mostly Firmicutes. In vivo assays allowed the identification of five candidate biocontrol strains against fusarium ear rot. Our data revealed novel insights into the role of the maize embryo microbiota and set the stage for further studies aimed at integrating this knowledge into plant breeding programs.

Occurrence of Toxigenic Fungi and Aflatoxin Potential of Aspergillus spp. Strains Associated with Subsistence Farmed Crops in Haiti.

Subsistence farming and poor storage facilities favor toxigenic fungal contamination and mycotoxin accumulation in staple foods from tropical countries such as Haiti. The present preliminary study was designed to evaluate the occurrence of toxigenic fungi in Haitian foodstuffs to define the mycotoxin risk associated with Haitian crops. The objectives of this research were to determine the distribution of toxigenic fungi in the Haitian crops maize, moringa, and peanut seeds and to screen Aspergillus section Flavi (ASF) isolates for production of aflatoxins B1 and G1 in vitro. Maize, moringa, and peanut samples were contaminated by potential toxigenic fungal taxa, mainly ASF and Fusarium spp. The isolation frequency of Aspergillus spp. and Fusarium spp. was influenced by locality and thus by farming systems, storage systems, and weather conditions. Particularly for ASF in peanut and maize samples, isolation frequencies were directly related to the growing season length. The present study represents the first report of contamination by toxigenic fungi and aflatoxin in moringa seeds, posing concerns about the safety of these seeds, which people in Haiti commonly consume. Most (80%) of the Haitian ASF strains were capable of producing aflatoxins, indicating that Haitian conditions clearly favor the colonization of toxigenic ASF strains over atoxigenic strains. ASF strains producing both aflatoxins B1 and G1 were found. Understanding the distribution of toxigenic ASF in Haitian crops and foodstuffs is important for determining accurate toxicological risks because the toxic profile of ASF is species specific. The occurrence of toxigenic fungi and the profiles of the ASF found in various crops highlight the need to prevent formation of aflatoxins in Haitian crops. This study provides relevant preliminary baseline data for guiding the development of legislation regulating the quality and safety of crops in this low-income country.

Competition assays revealed Paenibacillus pasadenensis strain R16 as a novel antifungal agent.

The development of new sustainable containment strategies of plant diseases is very important to guarantee food security while reducing the environmental impact of agriculture. Research of new biocontrol agents is a long and difficult process that involves several steps that start from the identification of possible candidates which, for example, show antibiotic activities, and ends with in field, large scale trials. In this study, the plant growth promoting potential and antifungal effect exerted by a novel, putative candidate biocontrol agent, strain R16, identified as Paenibacillus pasadenensis by sequence analysis of 16S rRNA and rpoB genes, against three important plant pathogenic fungi (Botrytis cinerea, Fusarium verticillioides, and Phomopsis viticola), were assessed. Biochemical assays to determine plant growth promoting potential gave negative results for siderophore production and phosphate solubilization, and positive results for ACC-deamination and IAA production. Further biochemical assays for endophytic lifestyle and antifungal activity gave positive results for catalase and chitinase activity, respectively. In vitro antagonism assays showed that strain R16 is effective against B. cinerea, reducing mycelial growth both in dual-culture and through volatile substances, characterized to be mostly composed by farnesol, and inhibiting conidia germination. Good antagonistic potential was also observed in vitro towards P. viticola, but not towards F. verticillioides. Moreover, in vivo assays confirmed the strain R16 activity reduced the infection rate on B. cinerea-inoculated berries. The obtained results firstly proved that P. pasadenesis strain R16 is a putative plant growth promoter and effective against phytopathogenic fungi. Further studies will be needed to investigate the possible application of this strain as a biocontrol agent.

Assessing pigmented pericarp of maize kernels as possible source of resistance to fusarium ear rot, Fusarium spp. infection and fumonisin accumulation.

One of the purposes of maize genetic improvement is the research of genotypes resistant to fusarium ear rot (FER) and fumonisin accumulation. Flavonoids in the pericarp of the kernels are considered particularly able to reduce the fumonisin accumulation (FUM). The aim of this field study was to assess the effect of flavonoids, associated with anti-insect protection and Fusarium verticillioides inoculation, on FER symptoms and fumonisin contamination in maize kernels. Two isogenic hybrids, one having pigmentation in the pericarp (P1-rr) and the other without it (P1-wr), were compared. P1-rr showed lower values of FER symptoms and FUM contamination than P1-wr only if the anti-insect protection and the F. verticillioides inoculations were applied in combination. Fusarium spp. kernel infection was not influenced by the presence of flavonoids in the pericarp. Artificial F. verticillioides inoculation was more effective than anti-insect protection in enhancing the inhibition activity of flavonoids toward FUM contamination. The interactions between FUM contamination levels and FER ratings were better modeled in the pigmented hybrid than in the unpigmented one. The variable role that the pigment played in kernel defense against FER and FUM indicates that flavonoids alone may not be completely effective in the resistance of fumonisin contamination in maize.

Identification and Characterization of New 'Candidatus Phytoplasma solani' Strains Associated with Bois Noir Disease in Vitis vinifera L. Cultivars Showing a Range of Symptom Severity in Georgia, the Caucasus Region.

Evidence from a preliminary survey highlighted that 'Candidatus Phytoplasma solani', the etiological agent of bois noir (BN) disease of grapevine, infects grapevine varieties in Georgia, a country of the South Caucasus. In this study, field surveys were carried out to investigate the BN symptom severity in international and Georgian native varieties. 'Ca. P. solani' was detected and identified by polymerase chain reaction-based amplification and restriction fragment length polymorphism analysis of 16S ribosomal DNA, and further characterized by multiple gene typing analysis (vmp1 and stamp genes). Obtained data highlighted that the majority of Georgian grapevine varieties showed moderate and mild symptoms, whereas international cultivars exhibited severe symptoms. Molecular characterization of 'Ca. P. solani' from grapevine revealed the presence of 11 distinct phytoplasma types. Only one type (VmGe12/StGe7) was identical to a strain previously reported in periwinkle from Lebanon; the other 'Ca. P. solani' types are described here for the first time. Phylogenetic analyses of vmp1 and stamp gene concatenated nucleotide sequences showed that 'Ca. P. solani' strains in Georgia are associated mainly with the bindweed-related BN host system. Moreover, the fact that 'Ca. P. solani' strains are distributed in grapevine cultivars showing a range of symptom intensity suggests a different susceptibility of such local cultivars to BN.

Sensitivity to cymoxanil in Italian populations of Plasmopara viticola oospores.

BACKGROUND: The level of sensibility towards cymoxanil was quantified by oospore germination assays in 28 populations of Plasmopara viticola (Berk. et Curt.) Berlese and De Toni sampled from different Italian regions from 2009 to 2012. RESULTS: The populations showed good sensitivity levels, with EC50 values often lower than 10 mg AI L(-1) and percentages of resistant individuals lower than 16%. Only three populations, sampled at the end of the 2012 grapevine growing season, were characterised by high resistance levels. Field trials carried out in two of these vineyards showed that, at the beginning of the 2013 grapevine growing season, the EC50 values of P. viticola populations as measured in the sporangial assay were higher than those observed with oospores. At the end of the season, in plots where cymoxanil was not applied, the populations fully reverted to sensitivity, while the EC50 values remained high where 3-6 applications were performed. CONCLUSION: Oospore germination assays provide valuable information on the sensitivity of populations in vineyards also at the quantitative level. The results obtained during the grapevine growing season confirm those obtained on the oospores, and that cymoxanil resistance is unstable, indirectly suggesting that the application of the fungicide according to antiresistance strategies can lead to good disease control.

The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence.

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre-formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post-extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual-step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties.

BACKGROUND: Grapevine downy mildew, caused by Plasmopara viticola, is a very serious disease affecting mainly Vitis vinifera cultivated varieties around the world. Breeding for resistance through the crossing with less susceptible species is one of the possible means to reduce the disease incidence and the application of fungicides. The hybrid Bianca and some of its siblings are considered very promising but their resistance level can vary depending on the pathogen strain. Moreover, virulent strains characterized by high fitness can represent a potential threat to the hybrid cultivation. RESULTS: The host response and the pathogen virulence were quantitatively assessed by artificially inoculating cv Chardonnay, cv Bianca and their siblings with P. viticola isolates derived from single germinating oospores collected in various Italian viticultural areas. The host phenotypes were classified as susceptible, intermediate and resistant, according to the Area Under the Disease Progress Curve caused by the inoculated strain. Host responses in cv Bianca and its siblings significantly varied depending on the P. viticola isolates, which in turn differed in their virulence levels. The fitness of the most virulent strain did not significantly vary on the different hybrids including Bianca in comparison with the susceptible cv Chardonnay, suggesting that no costs are associated with virulence. Among the individual fitness components, only sporangia production was significantly reduced in cv Bianca and in some hybrids. Comparative histological analysis revealed differences between susceptible and resistant plants in the pathogen diffusion and cytology from 48 h after inoculation onwards. Defence mechanisms included callose depositions in the infected stomata, increase in peroxidase activity, synthesis of phenolic compounds and flavonoids and the necrosis of stomata and cells immediately surrounding the point of invasion and determined alterations in the size of the infected areas and in the number of sporangia differentiated. CONCLUSIONS: Some hybrids were able to maintain an intermediate-resistant behaviour even when inoculated with the most virulent strain. Such hybrids should be considered for further field trials.

Mating behavior of a Northern Italian population of Fusarium verticillioides associated with maize.

Fusarium verticillioides, the most common causal organism of Fusarium stalk and ear rot of maize in Northern Italy, produces important mycotoxins such as fumonisins. Reproductive biology of F. verticillioides has been widely studied in numerous maize growing areas, but up to now no information is available on the mating behavior and genetic structure of this plant pathogen in Italy. Mating type and female fertility distribution and effective population number, N ( e ), were assessed for a population of 181 F. verticillioides strains isolated from three fields located in Lombardia region (Northern Italy) during 2007-2008 maize growing season. The ratio of MAT-1:MAT-2 was significantly different from the theoretical 1:1 ratio expected in an idealized population in which individuals mate at random. The frequency of hermaphroditic strains was 20 % of the total population. N ( e ) for mating type was 89 % of the count (total population) and the N ( e ) for male or hermaphrodite status was 55 %. The number of isolates that can function as the female parent limited N ( e ) in the examined population. Under equilibrium cycle, assuming that female fertility has been lost due to selection and mutation rate during asexual reproduction, sexual reproduction needed to occur only once per 40 to 118 asexual generations to maintain this level of sexual fertility.