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ABSTRACT Rhizopus microsporusis a necrotrophic post-harvest pathogen that causes significant economic losses in the agricultural sector. To explore alternatives to conventional management strategies for the mitigation of post-harvest infections, we investigated the potential of two previously identified endophyticBacillus velezensisstrains as biological control agents. Throughin vitroandin vivoexperiments, we examined the mechanisms of biocontrol displayed by twoB. velezensisstrains (KV10 and KV15) against threeR. microsporusstrains (W2-50, W2-51, and W2-58).In vitroassays assessed co-cultivability and the inhibitory effects ofB. velezensisagainstR. microsporus. The results demonstrated strain-specific antifungal activity with a reduction in fungal growth across treatments. Further analysis revealed that volatile organic compounds produced byB. velezensiscontributed to its antifungal properties. To evaluate the biocontrol efficacyin vivo, tomato fruits were inoculated withR. microsporusand subsequently treated withB. velezensis. The results support the strain-specific reduction in tomato spoilage, yielding various spoilage rates observed across treatments. Our findings highlight the potential ofB. velezensisas a promising biocontrol agent for the management ofR. microsporuspost-harvest infections in tomatoes. Further research is warranted to optimize the applicationof B. velezensisas a sustainable and environmentally friendly approach for controlling post-harvest diseases in tomatoes.IMPORTANCEOur study shows the significance of improving sustainable agriculture by offering an alternative to the use of chemical fungicides in post-harvest applications. Opportunistic fungal pathogens likeRhizopus microsporuscan have detrimental effects on post-harvest commodities like tomatoes. Post-harvest fungal infections are mainly controlled by chemical fungicides that pose health risks to humans and the environment. Utilizing biocontrol agents provides an environmentally safe alternative. Understanding the mechanisms of biocontrol employed by beneficial bacteria likeBacillus velezensison fungal pathogens gives insight into safer, more environmentally friendly alternatives to protect food crops. Our results suggest that targeted microbial solutions can mitigate post-harvest losses. 

Rhizopus rot is considered one of the most common diseases influencing global production and yield of horticulture commodities. However, the factors contributing to this pattern of prevalence are uncertain. Here, we focused on R. microsporus, which is known to rely on its endosymbiotic bacterium, Mycetohabitans, to produce toxins that interfere with plant development and inhibit the growth of other fungi. We assessed the impact of the symbiotic R. microsporus harboring its endosymbiont as well as the fungus cured of it on: (1) the magnitude of spoilage in tomato fruits, as evaluated by Koch's postulate for pathogenicity, (2) the shifts in native communities of endophytic fungi inhabiting these fruits, as examined by ITS rRNA gene metabarcoding and (3) secondary metabolites generated by these communities, as analyzed using multi-analyte LC-MS/MS. The pathogenicity test showed that the symbiotic endobacterium-containing R. microsporus W2-50 was able to cause tomato fruit spoilage. This was accompanied by decreased relative abundance of Alternaria spp. and an increase in the relative abundance of Penicillium spp. that may have facilitated the observed spoilage. In conclusion, symbiotic W2-50 appeared to facilitate fruit spoilage, possibly through successful colonization or toxin production by its endosymbiont. 

Abstract BackgroundTruffles are subterranean fungal fruiting bodies that are highly prized for their culinary value. Cultivation of truffles was pioneered in Europe and has been successfully adapted in temperate regions throughout the globe. Truffle orchards have been established in North America since the 1980s, and while some are productive, there are still many challenges that must be overcome to develop a viable North American truffle industry. These challenges include extended delays between establishment and production, comparatively low yields, high spatial heterogeneity in yield distribution, and orchard contamination with lower-value truffle fungi. AimHere we review known requirements for truffle production including necessary environmental conditions, reproductive biology, and effective agronomic practices. ContentWe consider the potential limitations of importing exotic host-fungal associations into North America where there is already a rich community of competing ectomycorrhizal fungi, host pests and pathogens. We also describe the status of the North American truffle industry with respect to market potential, including production costs, pricing, and biological and socioeconomic risk factors. A critical aspect of modern trufficulture involves monitoring with genetic tools that supply information on identity, abundance and distribution of fungal symbionts, abundance of competitive and contaminating fungi, and insight into the interactions between fungal mating types that are fundamental to the formation of truffle primordia. ImplicationsCultivation of the ectomycorrhizal truffle symbiosis requires application of pragmatic agronomic practices, adopting rigorous quality control standards, and an understanding of fungal biology, microbiology, and molecular biology. Consequently, significant interdisciplinary collaboration is crucial to further develop the North American truffle industry. 

EndofungalMycetohabitans(formerlyBurkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus,Rhizopus microsporus. The one known secreted effector family fromMycetohabitansconsists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenicXanthomonasandRalstoniaspp. to activate host genes that promote disease. These ‘BurkholderiaTAL-like (Btl)’ proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of twoMycetohabitansstrains revealed that the single Btl in oneMycetohabitans endofungorumstrain enhances fungal membrane stress tolerance, while others in aMycetohabitans rhizoxinicastrain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nineMycetohabitansspp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal–bacterial metagenomes. We show thatbtlgenes are present across diverseMycetohabitansstrains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested thatMycetohabitansmight contain more species than previously recognized. Within our data set, Btl proteins were more conserved acrossM. rhizoxinicastrains than acrossM. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial–fungal symbioses in myriad ways.