Plant Disease

Beyond the significant impact of Cassava witches broom disease (CWBD), caused by the fungus Rhizoctonia (syn. Ceratobasidium) theobromae on cassava cultivation in French Guiana and Brazil, this disease also poses a potential threat to cacao trees in the region, since the fungus is responsible for Vascular Streak Dieback (VSD) of cacao in South East Asia. Cross-pathogenicity trials were conducted in several cassava fields in French Guiana by planting young cacao plants adjacent to diseased cassava plants. Vascular necrosis was observed in some cacao plants, and the presence of R. theobromae in the cacao tissues was confirmed through PCR diagnostics using primers specific to the fungus. Sequence analysis indicated 100% similarity between samples from both hosts and 97.53 to 99.74% identity with R. theobromae isolates previously reported from cassava in the Americas and Southeast Asia. Additionally, symptomatic cacao in a mixed cacao-cassava farm yielded R. theobromae-positive PCR results, suggesting a natural infection. Ongoing work includes artificial inoculations and controlled cross-pathogenicity trials under screenhouse conditions to attempt reproduction of the symptoms. While current data do not yet establish definitive causality, the findings indicate potential host jump and warrant rapid communication to researchers, policy makers, and farmers to safeguard cacao production and Theobroma biodiversity in the Amazon region.

Cucurbit yellow vine disease (CYVD), caused by the bacterium Serratia ureilytica, is a phloem-associated disease of cucurbits. This study characterized the spatial and temporal distribution of S. ureilytica in Cucurbita pepo cultivar Delicata plants under greenhouse conditions using a GFP-tagged isolate (P01). Seedlings were sampled weekly for four weeks. Transverse sections from the stem, petiole, leaf, shoot apex, and root were imaged by laser scanning confocal and fluorescent dissecting microscopy. In parallel, bacterial abundance in each plant tissue was assessed by quantifying colony-forming units (CFU) via droplet plating over a 4-week time course. Across plant tissues and time points, S. ureilytica fluorescent signal was primarily concentrated in the inner and outer periphery of the bicollateral vascular bundles, with higher magnification images revealing mainly symplastic localization within phloem-associated cells. Consistent with the imaging results, bacterial quantification data showed a high abundance of CFUs in the main stem across weeks, with an irregular pattern of presence in the distal tissues at later time points. These results suggest that S. ureilytica is predominantly localized within phloem-associated cells and spreads both acropetally and basipetally during infection.

Australia is the largest producer of Pyrethrum (Tanacetum cinerariifolium) globally. Amongst the constraints on production are the fungal pathogens Didymella tanaceti and Stagonosporopsis tanaceti, which pose a significant threat to the industry, causing substantial yield losses. While the infection biology of S. tanaceti is well characterised, knowledge of D. tanaceti and its potential interaction with S. tanaceti on plants remains limited, hindering disease management. We developed fluorescently labelled strains of both pathogens via Agrobacterium tumefaciens-mediated transformation (ATMT). Binary vectors carrying the mNeonGreen or tdTomato fluorescent protein genes were introduced into D. tanaceti and S. tanaceti, respectively, and expression of the fluorescent proteins was confirmed by microscopy. Genome sequencing revealed single-copy T-DNA insertions in all transformants, with minor genomic rearrangements at insertion sites. Detached leaf assays demonstrated that transformed strains retained pathogenicity, producing disease symptoms indistinguishable from those of the wild type. These fluorescently labelled variants enabled detailed visualisation of D. tanaceti infection biology and its interactions with S. tanaceti, including co-infection dynamics. Co-infection assays using fluorescent strains further facilitated simultaneous visualisation and differentiation of both pathogens within host tissues. Importantly, these tools also allowed the first description of the early stages of infection by D. tanaceti in pyrethrum leaves. This study represents the first successful transformation of D. tanaceti and S. tanaceti, providing valuable resources to investigate their infection processes.

The fastidious basidiomycete Rhizoctonia (Ceratobasidium) theobromae is a biotrophic pathogen that causes Vascular-Streak Dieback (VSD) of Theobroma cacao (cocoa). The fungus has also been identified as the cause of an emergent disease known as Cassava Witches Broom Disease (CWBD) raising concerns that the pathogen is spreading to alternative hosts and to new regions. Interestingly, while VSD of cocoa and CWBD are reported as co-present in several countries, there is currently no evidence for cross-infection between species. The fungus is difficult to culture in vitro due its slow growth and Kochs postulates have not been definitive on either host. The complete fungus life cycle therefore remains enigmatic, though studies have progressed knowledge on pathology within the both the cocoa and cassava hosts. We have conducted limited field trials and sequenced mating (MAT) and ITS loci of isolates from various infected hosts and regions. We hypothesize that (i) genetic variation at MAT loci correlates with region or host (ii) long amplicon ITS sequences between isolates are more definitive for polymorphisms (iii) life-cycle traits of R. theobromae may be inferred from MAT loci (iv) cassava grown under VSD infected cocoa will be infected and develop symptoms of CWBD. We did not find any cross-infection in field trials, and we show that the pathogen is highly homozygous, despite undergoing meiosis, indicating a predominantly homothallic life cycle. Our data indicate that the pathogen is likely host specific and regionally divergent and suggests that host specificity on cocoa and cassava evolved by selection from a common ancestor rather than a host jump.

Summer squash (Cucurbita pepo) is a popular vegetable in Mississippi. These are harvested during the tender and immature stages. This vegetable is known to be a good source of vitamins A and C, as well as potassium, and iron. Small farms typically sell summer squash directly to consumers through local farmers markets. In this study, we isolated and identified a soft rot causing bacteria, Pectobacterium brasiliense strain 25ASUB12 (GenBank: PX884501), from symptomatic fruit of field-grown summer squash in Mississippi. We deployed both phenotypic and molecular techniques to identify this important pathogen which has a wide host range, including cucumbers, potatoes, and tomatoes.

Developing grapevine cultivars with genetic resistance to pathogens is a key strategy to reduce fungicide use and enhance sustainability. The French INRAE-ResDur program aims to pyramid several resistance loci against Plasmopara viticola (Rpv), the causal agent of downy mildew, while integrating factors against Erysiphe necator (Ren/Run) which causes powdery mildew. We evaluated in field the performance of grapevine genotypes carrying single or pyramided Rpv loci during the exceptionally severe downy mildew epidemic of 2024. Disease severity was quantified as the proportion of leaf foliage exhibiting symptoms. Susceptible controls averaged 66.6 % symptomatic leaves, Rpv1/3.1 combination remained below 16.1 %. whereas the Rpv1/Rpv3.1/Rpv10 pyramid showed only 4.9 % symptomatic leaves. The single loci provided partial protection, but the effect varied with genetic background. Pyramiding improved resistance effectiveness and stability, indicating synergistic interactions among loci. These findings demonstrate that pyramiding Rpv loci is an effective strategy for durable downy mildew resistance and should be the preferred strategy in grapevine breeding programs and genetic resistance deployment strategies.

Leaf diseases pose a serious threat to faba bean production. Leaf blotch of faba bean, caused by Alternaria spp., has become increasingly widespread and destructive in several countries. Leaf diseases pose a serious threat to faba bean production. The infection of plant by pathogens can be influenced by various factors associated with the host plant, environmental conditions and presence of other microorganisms. The phyllosphere and endosphere play a critical role in plant health and disease development. This study aimed to evaluate the factors shaping the structure and diversity of fungal communities associated with faba beans. Plant samples were collected in 2004 from two intensively managed faba bean production fields in the central region of Latvia. Fungal assemblages were characterized using an ITS region metabarcoding approach based on Illumina MiSeq sequencing. Among the assigned amplicon sequence variant (AVS), 65% belonged to the phylum Ascomycota, while approximately 4% were classified as Basidiomycota. Alternaria and Cladosporium were the dominant genera across samples. The alfa and beta diversities of fungal communities was higher during flowering of faba beans to compare with ripening. The higher abundance of Basidiomycota yeasts were observed during flowering, in contrast, Cladosporium genus was significantly more abundant during ripening. Alternaria DNA was found on leaves that showed no symptoms of the disease. The diversity and composition of fungal communities were significantly influenced by sampling time and presence of leaf blotch, caused by Alternaria spp.

Blackleg disease in potato, caused by soft rot Pectobacteriaceae, is a substantial cause of loss in seed potato production. Recent research has attempted to identify bacteria with antagonistic activity against several diseases, among which blackleg. However, most biocontrol agents have been tested only in-vitro or in the greenhouse. In this study, we tested the effect of bacterial biocontrol agents in a four-year field experiment against blackleg caused by Pectobacterium brasiliense and Dickeya solani. Effects of the treatments on disease incidence was highly variable between years and also differed between cultivars, soil type and even replicates. Disease incidence was on average higher in sandy soil compared to clay soil and higher in the cultivar Kondor than Mozart. For a subset of the bacterial isolates genome mining could detect the presence of genes involved in the production of antibiotics and siderophores, but this was not correlated with disease incidence in the field. Moreover, most isolates were able to survive in storage on tubers from inoculation until planting. Thus, we conclude that while the used isolates showed the potential for antagonistic activity and were present on tubers when planting, no antagonist treatment could consistently decrease disease incidence. Inoculation of the isolates on the tuber surface might have been insufficient for plant colonization.

Wild potatoes are long-evolving relatives of the cultivated potatoes we have today. These wild Solanum species harbor traits that can be exploited to develop more nutritive and resilient potato varieties, providing the genetic basis for resistance to abiotic and biotic stresses such as drought, low temperatures, diseases and pests. Wild potato species are widely used as valuable genetic resources in breeding programs, including efforts aimed at improving resistance to bacterial wilt caused by Ralstonia solanacearum. Among the wild species native to Uruguay, Solanum malmeanum has emerged as a particularly valuable source of resistance. The aim of this work was to investigate weather differences in bacterial wilt resistance among S. malmeanum accessions are associated with structural and compositional changes of rhizosphere bacterial communities. Two S. malmeanum accessions were compared, one susceptible (RN9P2) and the other resistant (A11P1) to bacterial wilt. The impact of plant resistance and pathogen colonization on the structure of rhizosphere bacterial communities were evaluated using high throughput 16S rRNA gene amplicon-sequencing. Significant differences were observed between accessions and pronounced shifts in rhizosphere bacterial communities were detected in response to pathogen inoculation. Cryseobacterium, Sphingobacterium, Komagataeibacter, Gluconobacter, Lactobacillus and Dyella were differential genera and enriched in the rhizosphere of the resistant accession. Several of these genera have been previously associated with disease suppression. Overall, these results suggest that the rhizosphere bacterial community associated with resistant S. malmeanum accessions may contribute to protection against R. solanacearum infection.

Host resistance is a critical component of oat crown rust disease management. Pc94 is a qualitative resistance locus derived from diploid Avena strigosa with several independent introgressions into A. sativa that have been used in cultivar deployment. Quantitative trait locus (QTL) analysis combining previously published data for a historic A. strigosa population segregating for Pc94 revealed a large effect QTL on the distal end of A. strigosa chromosome 7A. Genome assembly of the parents identified a cluster of five nucleotide binding site leucine-rich repeat receptor (NLR) candidate genes within the QTL region. A single candidate NLR with an integrated zinc finger BED domain, AstNLR94, was determined as necessary for Pc94 resistance based on map-based cloning and forward mutagenesis. A presence/absence allele specific PCR marker was designed in AstNLR94 and verified for accuracy and specificity in a diverse panel of A. strigosa and A. sativa. Pc94 introgressions in A. sativa ranged in size from 1.7-71 Mbp and two different introgression locations appear to have occurred. In A. sativa Leggett, a 6.3 Mbp Pc94 introgression is located at the end of chromosome 2A, and the same sized introgression was discovered in the OT3098 v2 genome. Finally, a QTL analysis identified an additional minor resistance locus on A. strigosa chromosome 4A, which has complicated previous efforts to characterize the Pc94 locus. This is the first report of an NLR gene underlying disease resistance in Avena spp. and delivers a Pc94 marker for marker assisted selection to produce disease resistant cultivars. Key messageWe mapped a zfBED-NLR encoding gene necessary for Pc94 resistance, developed a diagnostic marker, and revealed diverse introgression sizes, clarifying Pc94s history and utility for durable oat crown rust resistance.

The rapid expansion of oil palm plantations in Southeast Asia has caused extensive deforestation and landscape fragmentation. Riparian buffers (vegetated strips along the edges of rivers) have been shown to enhance biodiversity, water quality, and erosion control. However, plantation managers have raised concerns that these buffers may harbour pests such as nettle caterpillars, bagworms, and rhinoceros beetles (Oryctes rhinoceros). These pests damage the palms and facilitate the spread Ganoderma boninense (a fungal pathogen). Using causal inference modelling we examined how riparian buffer characteristics (width and habitat quality), oil palm age, and surrounding landscape features influence pest and disease incidence in oil palms adjacent to riparian areas in Sabah, Malaysian Borneo. We surveyed 47,500 palms for pest and disease damage and used mark-release-recapture techniques to track O. rhinoceros movements in oil palms adjacent to riparian buffers. Most O. rhinoceros activity (66.30%) occurred within the plantations, and only 6.10% occurred within riparian buffers, with limited movement between habitats. Oil palm age was a dominant driver of pest attacks: young palms were more susceptible to lepidopteran caterpillars and O. rhinoceros, whereas G. boninense was more prevalent in mature palms. Neither the surrounding forest cover nor the quality of the riparian buffer affected the incidence of pest attacks. Riparian buffer width increased O. rhinoceros attacks, reduced G. boninense infection, and had no effect on lepidopteran caterpillars, highlighting that surrounding forest cover and riparian buffers do not drive pest attacks in oil palm plantations. Instead, management of oil palms within the buffers s is likely to be more important in managing pests; increases in invasive oil palms within the buffers increased the incidence of caterpillar damage, and higher numbers of remnant old oil palms increased O. rhinoceros attacks in adjacent oil palms. Overall, riparian buffers were found to contribute little to pest spillover, suggesting that their biodiversity and connectivity benefits outweigh minor pest risks, especially if invasive young and remnant old oil palms within the buffers are effectively managed and native vegetation restored.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other Brassica crops. Polymerase chain reaction (PCR), particularly probe-based quantitative PCR (qPCR), is widely used for the detection of P. brassicae in soil samples. To improve consistency in clubroot detection while maintaining efficiency, diagnostic laboratories would benefit from adopting a single, highly efficient qPCR system for routine testing. In this study, we analyzed the primer and probe sequences of all published PCR and qPCR systems for P. brassicae detection. Based on these analyses, three independently developed probe-based qPCR systems were selected and their performance was evaluated using synthesized target DNA (gBlock). One probe-based qPCR system exhibiting superior sensitivity on gBlock was subsequently evaluated on P. brassicae genomic DNA. This system consistently detected DNA equivalent to four resting spores per reaction, corresponding to a soil sample containing 1,000 spores per g soil when the DNA extraction protocol was considered as a component of the qPCR system. The sensitivity of the system was further validated using DNA extracted from soil samples collected from multiple locations across Alberta, where P. brassicae was detected at levels below those associated with visible clubroot symptoms. Based on these results, we recommend this qPCR system for routine clubroot diagnostics in laboratories across Canada.

BackgroundPotato is an important crop worldwide, yet its production is severely threatened by Phytophthora infestans, the causal agent of late blight. Alternatives to the current control strategies are needed, as these rely heavily on environmentally harmful treatments. The recruitment of beneficial microbes by plants upon stress ("cry-for-help" mechanism) may represent an opportunity to find new biocontrol agents but this has not yet been reported for potato. The aim of this study was to analyse whether foliar late blight infection induces shifts in the phyllosphere, rhizosphere and soil bacterial communities associated with two potato cultivars of differing sensitivity to late blight. Moreover, we aimed at isolating members of the plant microbiota to test whether bacteria putatively recruited upon infection would be particularly active in protecting the plant against late blight. ResultsControlled foliar infection triggered substantial, cultivar-specific shifts in the rhizosphere communities across two successive generations. Despite the number of differentially abundant ASVs detected being ten times higher in the second generation than in the first one, the same taxonomic groups were concerned by the shifts: Burkholderiales, Flavobacteriales, and Bacillales. Furthermore, the communities linked to the susceptible cultivar consistently shifted more strongly than the communities linked to the resistant cultivar. The obtained ASV sequences were used to identify 163 corresponding isolates. The inhibition potential of these strains against P. infestans spores was assessed through biological assays, which revealed the biocontrol potential of strains otherwise not yet known to inhibit phytopathogenic organisms, such as Advenella, Nocardioides and Phyllobacterium strains. Although we found no correlation between the relative abundance shift of the ASVs upon infection and the activity of the corresponding strains, we observed that the overall activity of strains isolated from the resistant cultivar was higher than that of the strains isolated from the susceptible one. ConclusionTaken together, the higher activity of the strains isolated from the resistant cultivar, along with its comparatively modest microbiome shifts upon infection suggest that the investigated resistant cultivar might harbour specific microbiota enriched in strains with efficient protective abilities against their host plants pathogens, which possibly contribute to its higher resistance against P. infestans.

Banana leaf diseases are a significant threat to Cavendish banana production. In the Philippines, the main disease has been diagnosed as Black sigatoka disease caused by Pseudocercospora fijiensis based on symptoms. However, our study showed that the main pathogen in Mindanao island, the largest banana-producing region in the Philippines, belongs to the genus Nigrospora, contradicting previous assumptions. We clarified the phylogenetic positions of 160 Nigrospora isolates based on molecular phylogenetic analyses using ITS, {beta}-tubulin, and tef1 sequences, and compared their morphology with known species. Molecular phylogenetic and morphological analysis revealed that Nigrospora isolates comprised N. chinensis, N. lacticolonia, N. cf. singularis, N. sphaerica, N. vesicularifera, and a novel species, N. nigrocolonia. Pathogenicity tests on banana leaves confirmed that these species are pathogenic. Species other than N. sphaerica were for the first time reported as pathogens of banana leaf. The results of the fungicide sensitivity test using 14 fungicides, including pyrimethanil, spiroxamine, and tebuconazole, for the Sigatoka disease showed 100% inhibition of all isolates at 100 ppm of active ingredients. However, low-sensitivity isolates were observed for the remaining 11 fungicides. Our findings indicated the need for a comprehensive review of banana leaf disease prevention strategies.

The objective of this study was to re-analyse the molecular phylogeny and/or the morphology of all species, which have been attributed to the so-far mono-generic fungal family Ambisporaceae. The genus Ambispora has been well-known for its spore bi-morphy described even from single spore clusters. Triple-walled spores are differentiated on sporiferous saccules, while mono-walled spores are formed on simple subtending hyphae. New phylogenetic analyses reveal dissimilarities of [≥]10% in partial nrDNA gene of three different stable phylogenetic clades and thus suggest the division of Ambispora into three genera, which simultaneously request for advanced morphological separations. These advances are primarily based on the more diverse spore wall composition of the ambisporoid-acaulosporoid morph rather than on the rather simple-glomoid morph. While all known species of the triple-walled morph have an evanescent to semi-permanent outer spore wall, i) Am. fennica, Am. brasiliensis, Am. gerdemannii and Am. nicolsonii have a smooth, permanent central spore wall (Am. fennica clade, A), ii) the central wall of Am. appendicula, Am. callosa, Am. leptoticha and Am. jimgerdemannii is alveolate (Am. appendicula clade, B), and iii) the central wall of Am. granatensis is smooth, but easily degraded, thus rather short-lived and not permanent but evanescent (Am. granatensis clade, C). In conclusion, species of the Am. fennica clade represent the genus Ambispora, while species of the Am. appendicula clade represent the new genus Appendiculaspora, and the mono-specific Am. granatensis clade represents the new genus Ephemerapareta. Species of an additional morph, with triple-walled spores, but apparently formed on subtending hyphae, and having a diagnostic reticulate, football-like middle wall, are here separated from the revised genus Ambispora based solely on morphological analyses, since molecular identification analyses so far failed and remained merely unknown. This later morph and genus is based on the type species Pelotaspora reticulata comb. nov, and on P. austrolatina sp. nov. Concomitant molecular phylogenetic and morphological analyses are needed to attribute not only Pelotaspora spp., but also those species, for which hitherto only the ambisporoid-glomoid morph has been observed correctly within the family Ambisporaceae. Without molecular analyses, such species with glomoid but unknown ambisporoid-acaulosporoid morph have to be retained within Ambispora.

Vertical transmission of plant pathogenic viruses is an important component of viral persistence, survival, and spread in agricultural production systems. This type of transmission is of considerable economic significance as it can cause major crop losses by serving as the initial focus of infection for future epidemics. Vertical transmission occurs when a virus is passed on to offspring either by direct invasion of the developing seed embryo from infected mother plants or through infected pollen grains after fertilization. We have recently demonstrated by high throughput sequencing that mature seeds of the agriculturally important forage crop alfalfa (Medicago sativa L.) are associated with a broad range of viruses some of which could potentially spread over long distances via seed. Aside from alfalfa mosaic virus, little is currently known about viral transmission via alfalfa pollen and its role in the epidemiology in this crop. This research was conducted to screen the pollen obtained from unique alfalfa genotypes for the presence of pathogenic viruses and their potential for dissemination. The plants from which the pollen was collected were alfalfa genotypes selected for fungal plant disease resistance and agronomic performance in a USDA ARS pre-breeding program in Prosser, WA.

Pyrenophora teres f. teres (Ptt), the causal agent of net form net blotch disease in barley, is an economically important fungal pathogen worldwide. Understanding both host resistance mechanisms and pathogen virulence factors is essential for developing durable net form net blotch resistant barley cultivars. Quantitative trait loci (QTL) mapping was conducted using a cross between two Ptt isolates, one virulent on the barley cultivar Prior and the other being avirulent. A major QTL associated with virulence on Prior was detected on chromosome 5. A progeny isolate possessing this QTL, together with the two parental isolates, was subsequently used in the proteomic analyses. Label-free proteomics was used to quantify in planta the protein profile changes in Prior following inoculations with the virulent and avirulent parental Ptt isolates, and the virulent progeny isolate. Leaf samples were collected at two (D2) and five (D5) days post-inoculation, and proteomic analyses performed to identify proteins associated with host resistance and pathogen virulence. A dataset comprising 2,886 barley proteins and 51 Ptt proteins was analysed. Principal component analysis (PCA) of the barley Prior proteomes revealed distinct clustering based on resistance and susceptibility at D5, while D2 samples formed a separate cluster. The PCA of the Ptt proteomes identified separate clusters, one comprised of the D2 and D5 avirulent parental isolate and another cluster of the virulent isolates at D5 only. Gene ontology analysis of the Prior proteins that were significantly increased in the resistant compared to the susceptible groups revealed functional categories related to protein translation, biosynthesis and chloroplast activities. The proteins that were significantly increased in the susceptible compared to the resistant Prior group were associated with organic acid and carbohydrate metabolism. The proteomic profiles and bioinformatic analysis generated in our study provide novel insights into the molecular basis of resistance and virulence in the barley-P. teres pathosystem. Key messageThis study reveals the first in planta proteomic profiles of both barley and Pyrenophora teres f. teres, identifying unique virulence-associated proteins and host responses linked to resistance and susceptibility.

Population genetic analysis of species of conservation concern provides information to devise management plans to effectively conserve the genetic variation of endangered species. One such endangered plant, Physaria globosa is a federally endangered species in the mustard family with a geographically restricted range that occurs in four disjunct locations in Indiana, Kentucky, and Tennessee (i.e., Highland Rim and Nashville Basin regions) and along the Wabash, Kentucky, and Cumberland Rivers. In this study, we sampled populations from throughout the range of P. globosa, genotyped them using 20 microsatellite loci, and assessed genetic diversity and structure within and among populations. The goals of the study were to understand: 1) levels of genetic diversity in P. globosa and whether populations show evidence of having experienced reductions in genetic diversity as the result of genetic bottlenecks, genetic drift, or inbreeding, 2) rangewide genetic diversity and structure in P. globosa and how genetic structure is affected by the disjunctions in the species range, and 3) implications for prioritization of in-situ and ex-situ conservation efforts. On average, P. globosa showed comparable levels of genetic diversity to other species of Physaria. However, some populations showed evidence of inbreeding, genetic bottlenecks, or decreases in genetic diversity, possibly due to anthropogenic or climate-related pressures and decreases in population size due to competition with invasive bush honeysuckle. Genetic variation was strongly structured into two main geographic groups, one in the northern part of the species range (KY and IN), and the other in the southern part of the species range (TN), but some populations likely originated via long-distance dispersal. We also found significant isolation by distance, likely due to both life history characteristics and physical barriers associated with the complex topological structure of the landscape occupied by P. globosa, limiting population connectivity. Given the strong genetic structure found in P. globosa, several populations should be protected and managed within each geographic region to conserve genetic variation. Ex situ conservation will also be important to protect genetic diversity, particularly for populations that are difficult to access and manage.

RNA interference (RNAi) shows great potential to protect crops against fungal diseases, yet reported protection efficiencies vary greatly, and our understanding of the factors responsible for this variance remains limited. In this meta-analysis, we evaluated 89 studies that compare the efficiency of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) in controlling fungal diseases, focusing on biotrophic, hemibiotrophic, and necrotrophic fungi, the use of formulations, and the dsRNA design as explanatory factors for differences between reported efficiency values. Our results indicate that SIGS is slightly more effective, particularly in biotrophs. Surprisingly, SIGS studies using formulations did not outperform those applying naked dsRNA. We also assessed parameters of RNA design. Differences in dsRNA length and the number of constructs, and number of targets showed no consistent significant effect on resistance in either HIGS or SIGS. Interestingly, however, HIGS studies reported significantly higher efficiency when targeting genes closer to the 3 end and SIGS when targeting genes closer to the 5 end. We discuss potential reasons for the reported patterns, such as variability in dsRNA uptake mechanisms, intercellular trafficking and Dicer processing, and conclude that more research is needed to understand the biological mechanisms determining RNAi efficiency for fungal control.

BackgroundThe environment in which a fungus grows can directly influence their development, transmission, and pathogenic potential. This environment encompasses factors like nutrient availability, biotic and abiotic stressors, as well as host-derived chemical cues. In fungal pathogens, where conidia act as the infectious agents, the environment impacts the quantity and quality of these spores, thereby aOecting their ability to infect and kill hosts. In the present study, we investigated the effect of host-derived medium types on various phenotypes, including spore production, growth rate, and virulence in two entomopathogenic fungi, Metarhizium acridum and Metarhizium brunneum. Three medium types derived from insect material were compared to a standard laboratory medium. ResultsConidia produced on the insect-derived media exhibited enhanced sporulation and reduced time to sporulation, while conidial germination and maximum growth rate were comparable across medium types, suggesting that some of the medium-induced phenotypic effects were transient. Notably, conidia derived from two of the insect medium types demonstrated higher virulence, indicating that host-derived cues may prime virulence. ConclusionThese results highlight that the composition of growth substrates can regulate fungal reproductive strategies and virulence, with implications for developing high-throughput phenotyping and for the biotechnological optimization of mass production and efficacy of entomopathogenic fungi in biological control applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/711814v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@189013eorg.highwire.dtl.DTLVardef@1b0cedborg.highwire.dtl.DTLVardef@dccb4eorg.highwire.dtl.DTLVardef@1a77895_HPS_FORMAT_FIGEXP M_FIG C_FIG