Inter-species interactions between two bacterial flower commensals and a floral pathogen reduce disease incidence and alter pathogen activity.

Flowers are colonized by a diverse community of microorganisms that can alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, members of the genera Pseudomonas and Pantoea, isolated from apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between the two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower commensal strains did not competitively exclude E. amylovora from the stigma habitat, as both bacteria and the pathogen co-existed on the stigma of apple flowers and in vitro. This suggests that plant protection might be mediated by other mechanisms than competitive niche exclusion. Using a synthetic stigma exudation medium, ternary co-culture of the bacterial strains led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Importantly, the gene expression profiles for the ternary co-culture were not just additive from binary co-cultures, suggesting that some functions only emerged in multipartite co-culture. Additionally, the ternary co-culture of the strains resulted in a stronger acidification of the growth milieu than mono- or binary co-cultures, pointing to another emergent property of co-inoculation. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their potential impact on plant health and pathogen behavior. IMPORTANCE: Fire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen's behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.

Microbe-mediated alterations in floral nectar: consequences for insect parasitoids.

Floral nectar is frequently colonized by microbes among which bacteria and yeasts are the most abundant. These microbes have the ability to alter nectar characteristics with consequences for the whole community of flower-visiting insects. Recent research carried out on natural enemies of insect herbivores has shown that microbe-mediated changes in nectar traits can influence the foraging behavior and life history traits of parasitoids. The production of microbial volatile organic compounds can affect the attraction of parasitoids to nectar, while changes in sugar and amino acid composition can impact their longevity. Future research should focus on understanding the effects of nectar microbial colonization on parasitoid reproduction, with a specific emphasis on the interactions among different microbial taxa known to co-occur in floral nectar. Overall, this review highlights the importance of considering the role of nectar-inhabiting microbes in shaping the interactions between parasitoids and their food resources.

Contribution of Native Plasmids of Pantoea vagans C9-1 to Epiphytic Fitness and Fire Blight Management on Apple and Pear Flowers and Fruits.

Pantoea vagans C9-1 (C9-1) is a biological control bacterium that is applied to apple and pear trees during bloom for suppression of fire blight, caused by Erwinia amylovora. Strain C9-1 has three megaplasmids: pPag1, pPag2, and pPag3. Prior bioinformatic studies predicted these megaplasmids have a role in environmental fitness and/or biocontrol efficacy. Plasmid pPag3 is part of the large Pantoea plasmid (LPP-1) group that is present in all Pantoea spp. and has been hypothesized to contribute to environmental colonization and persistence, while pPag2 is less common. We assessed fitness of C9-1 derivatives cured of pPag2 and/or pPag3 on pear and apple flowers and fruit in experimental orchards. We also assessed the ability of a C9-1 derivative lacking pPag3 to reduce populations of E. amylovora on flowers and disease incidence. Previously, we determined that tolerance to stresses imposed in vitro was compromised in derivatives of C9-1 lacking pPag2 and/or pPag3; however, in this study, the loss of pPag2 and/or pPag3 did not consistently reduce the fitness of C9-1 on flowers in orchards. Over the summer, pPag3 contributed to survival of C9-1 on developing apple and pear fruit in two of five trials, whereas loss of pPag2 did not significantly affect survival of C9-1. We also found that loss of pPag3 did not affect C9-1's ability to reduce E. amylovora populations or fire blight incidence on apple flowers. Our findings partially support prior hypotheses that LPP-1 in Pantoea species contributes to persistence on plant surfaces but questions whether LPP-1 facilitates host colonization.

Interspecies and temporal dynamics of bacterial and fungal microbiomes of pistil stigmas in flowers in holoparasitic plants of the Orobanche series Alsaticae (Orobanchaceae).

Little is known about the microbiomes of flower parts, and even less information is available regarding these microorganisms' colonization of specific niches in parasitic plants. We investigate the temporal interspecies dynamics of the parasitic plants microbiome of flower stigmas in two stages of development: immature stigmas in flower buds and mature stigmas in opened flowers. We compared two related holoparasitic Orobanche species from localities approximately 90 km apart and characterize their bacterial and fungal communities using 16S rRNA gene and ITS sequences, respectively. We identified from 127 to over 228 OTUs per sample for fungi, sequences belonging to genera: Aureobasidium, Cladosporium, Malassezia, Mycosphaerella, and Pleosporales, constituting approximately 53% of the community in total. In the bacterial profile, we recorded 40 to over 68 OTUs per sample consisting of Enterobacteriaceae, and genera Cellulosimicrobium, Pantoea, and Pseudomonas spp., with an approximately 75% frequency. In microbial communities, higher numbers of OTUs colonizing mature stigmas were recorded than in immature. This implies that the dynamics and concurrence of microbial communities were different between O. alsatica and O. bartlingii and underwent significant changes during flower development. To the best of our knowledge, is the first study of the interspecies and temporal dynamics of the bacterial and fungal microbiomes of pistil stigmas in flowers.

Inter- and intraspecific phytochemical variation correlate with epiphytic flower and leaf bacterial communities.

Microbes associated with flowers and leaves affect plant health and fitness and modify the chemical phenotypes of plants with consequences for interactions of plants with their environment. However, the drivers of bacterial communities colonizing above-ground parts of grassland plants in the field remain largely unknown. We therefore examined the relationships between phytochemistry and the epiphytic bacterial community composition of flowers and leaves of Ranunculus acris and Trifolium pratense. On 252 plant individuals, we characterized primary and specialized metabolites, that is, surface sugars, volatile organic compounds (VOCs), and metabolic fingerprints, as well as epiphytic flower and leaf bacterial communities. The genomic potential of bacterial colonizers concerning metabolic capacities was assessed using bacterial reference genomes. Phytochemical composition displayed pronounced variation within and between plant species and organs, which explained part of the variation in bacterial community composition. Correlation network analysis suggests strain-specific correlations with metabolites. Analysis of bacterial reference genomes revealed taxon-specific metabolic capabilities that corresponded with genes involved in glycolysis and adaptation to osmotic stress. Our results show relationships between phytochemistry and the flower and leaf bacterial microbiomes suggesting that plants provide chemical niches for distinct bacterial communities. In turn, bacteria may induce alterations in the plants' chemical phenotype. Thus, our study may stimulate further research on the mechanisms of trait-based community assembly in epiphytic bacteria.

Fructilactobacillus cliffordii sp. nov., Fructilactobacillus hinvesii sp. nov., Fructilactobacillus myrtifloralis sp. nov., Fructilactobacillus carniphilus sp. nov. and Fructobacillus americanaquae sp. nov., five novel lactic acid bacteria isolated from insects or flowers of Kangaroo Island, South Australia.

Six strains, KI11_D11T, KI4_B1, KI11_C11T, KI16_H9T, KI4_A6T and KI3_B9T, were isolated from insects and flowers on Kangaroo Island, South Australia. On the basis of 16S rRNA gene phylogeny, strains KI11_D11T, KI4_B1, KI11_C11T, KI16_H9T, KI4_A6T were found to be closely related to Fructilactobacillus ixorae Ru20-1T. Due to the lack of a whole genome sequence for this species, whole genome sequencing of Fructilactobacillus ixorae Ru20-1T was undertaken. KI3_B9T was found to be closely related to Fructobacillus tropaeoli F214-1T. Utilizing core gene phylogenetics and whole genome analyses, such as determination of AAI, ANI and dDDH, we propose that these six isolates represent five novel species with the names Fructilactobacillus cliffordii (KI11_D11T= LMG 32130T = NBRC 114988T), Fructilactobacillus hinvesii (KI11_C11T = LMG 32129T = NBRC 114987T), Fructilactobacillus myrtifloralis (KI16_H9T= LMG 32131T = NBRC 114989T) Fructilactobacillus carniphilus (KI4_A6T = LMG 32127T = NBRC 114985T) and Fructobacillus americanaquae (KI3_B9T = LMG 32124T = NBRC 114983T). Chemotaxonomic analyses detected no fructophilic characters for these strains of member of the genus Fructilactobacillus. KI3_B9T was found to be obligately fructophilic, similarly to its phylogenetic neighbours in the genus Fructobacillus. This study represents the first isolation, to our knowledge, of novel species in the family Lactobacillaceae from the Australian wild.

Nectar compounds impact bacterial and fungal growth and shift community dynamics in a nectar analog.

Floral nectar is frequently colonised by microbes. However, nectar microbial communities are typically species-poor and dominated by few cosmopolitan genera. One hypothesis is that nectar constituents may act as environmental filters. We tested how five non-sugar nectar compounds as well as elevated sugar impacted the growth of 12 fungal and bacterial species isolated from nectar, pollinators, and the environment. We hypothesised that nectar isolated microbes would have the least growth suppression. Additionally, to test if nectar compounds could affect the outcome of competition between microbes, we grew a subset of microbes in co-culture across a subset of treatments. We found that some compounds such as H2 O2 suppressed microbial growth across many but not all microbes tested. Other compounds were more specialised in the microbes they impacted. As hypothesised, the nectar specialist yeast Metschnikowia reukaufii was unaffected by most nectar compounds assayed. However, many non-nectar specialist microbes remained unaffected by nectar compounds thought to reduce microbial growth. Our results show that nectar chemistry can influence microbial communities but that microbe-specific responses to nectar compounds are common. Nectar chemistry also affected the outcome of species interactions among microbial taxa, suggesting that non-sugar compounds can affect microbial community assembly in flowers.

Sugar Concentration, Nitrogen Availability, and Phylogenetic Factors Determine the Ability of Acinetobacter spp. and Rosenbergiella spp. to Grow in Floral Nectar.

The floral nectar of angiosperms harbors a variety of microorganisms that depend predominantly on animal visitors for their dispersal. Although some members of the genus Acinetobacter and all currently known species of Rosenbergiella are thought to be adapted to thrive in nectar, there is limited information about the response of these bacteria to variation in the chemical characteristics of floral nectar. We investigated the growth performance of a diverse collection of Acinetobacter (n = 43) and Rosenbergiella (n = 45) isolates obtained from floral nectar and the digestive tract of flower-visiting bees in a set of 12 artificial nectars differing in sugar content (15% w/v or 50% w/v), nitrogen content (3.48/1.67 ppm or 348/167 ppm of total nitrogen/amino nitrogen), and sugar composition (only sucrose, 1/3 sucrose + 1/3 glucose + 1/3 fructose, or 1/2 glucose + 1/2 fructose). Growth was only observed in four of the 12 artificial nectars. Those containing elevated sugar concentration (50% w/v) and low nitrogen content (3.48/1.67 ppm) were limiting for bacterial growth. Furthermore, phylogenetic analyses revealed that the ability of the bacteria to grow in different types of nectar is highly conserved between closely related isolates and genotypes, but this conservatism rapidly vanishes deeper in phylogeny. Overall, these results demonstrate that the ability of Acinetobacter spp. and Rosenbergiella spp. to grow in floral nectar largely depends on nectar chemistry and bacterial phylogeny.

Physiological stage drives fungal community dynamics and diversity in Leptospermum scoparium (manuka) flowers.

Flowers are an important niche for microbes, and microbes in turn influence plant fitness. As flower morphology and biology change rapidly over time, dynamic niches for microbes are formed and lost. Floral physiology at each life stage can therefore influence arrival, persistence and loss of microbial species; however, this remains little understood despite its potential consequences for host reproductive success. Through internal transcribed spacer 1 (ITS1) community profiling, we characterized the effect of transitioning through five floral stages of manuka (Leptospermum scoparium), from immature bud to spent flower, and subsequent allocation to seed, on the flower-inhabiting fungal community. We found nectar-consuming yeasts from Aureobasidium and Vishniacozyma genera and functionally diverse filamentous fungi from the Cladosporium genus dominated the anthosphere. The candidate core microbiota persisted across this dynamic niche despite high microbial turnover, as observed in shifts in community composition and diversity as flowers matured and senesced. The results demonstrated that floral stages are strong drivers of anthosphere fungal community assembly and dynamics. This study represents the first detailed exploration of fungi through floral development, building on fundamental knowledge in microbial ecology of healthy flowers.

Overproduction of OsRACK1A, an effector-targeted scaffold protein promoting OsRBOHB-mediated ROS production, confers rice floral resistance to false smut disease without yield penalty.

Grain formation is fundamental for crop yield but is vulnerable to abiotic and biotic stresses. Rice grain production is threatened by the false smut fungus Ustilaginoidea virens, which specifically infects rice floral organs, disrupting fertilization and seed formation. However, little is known about the molecular mechanisms of the U. virens-rice interaction and the genetic basis of floral resistance. Here, we report that U. virens secretes a cytoplasmic effector, UvCBP1, to facilitate infection of rice flowers. Mechanistically, UvCBP1 interacts with the rice scaffold protein OsRACK1A and competes its interaction with the reduced nicotinamide adenine dinucleotide phosphate oxidase OsRBOHB, leading to inhibition of reactive oxygen species (ROS) production. Although the analysis of natural variation revealed no OsRACK1A variants that could avoid being targeted by UvCBP1, expression levels of OsRACK1A are correlated with field resistance against U. virens in rice germplasm. Overproduction of OsRACK1A restores the OsRACK1A-OsRBOHB association and promotes OsRBOHB phosphorylation to enhance ROS production, conferring rice floral resistance to U. virens without yield penalty. Taken together, our findings reveal a new pathogenic mechanism mediated by an essential effector from a flower-specific pathogen and provide a valuable genetic resource for balancing disease resistance and crop yield.

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) for Detection of Shiga Toxin Gene (stx1 and/or stx2) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071903.

BACKGROUND: The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) method is based on gene amplification by the use of real-time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) was approved as AOAC Performance Tested MethodSM Certificate No. 071903. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx) product instructions and Standard Method Performance Requirement (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx) results and the SMPR 2020.012 recommended cultural confirmations. CONCLUSIONS: This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method is suitable for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower.

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) for Detection of Shiga Toxin Gene (stx1 and/or stx2) and Intimin Gene (eae) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071902.

BACKGROUND: The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) and intimin gene (eae) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) was approved as AOAC Performance Tested MethodSM Certificate No. 071902. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) product instructions and Standard Method Performance Requirements (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) results and the SMPR 2020.012 recommended cultural confirmations. CONCLUSIONS: This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) is a reliable method for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is suitable for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower.

Priority Effects in the Apple Flower Determine If the Siderophore Desferrioxamine Is a Virulence Factor for Erwinia amylovora CFBP1430.

Iron is crucial for bacterial growth and virulence. Under iron-deficiency bacteria produce siderophores, iron chelators that facilitate the iron uptake into the cell via specific receptors. Erwinia amylovora, the causative agent of fire blight, produces hydroxamate-type desferrioxamine siderophores (DFO). The presented study reassesses the impact of DFO as a virulence factor of E. amylovora during its epiphytic phase on the apple flower. When inoculated in semisterile Golden Delicious flowers no difference in replication and induction of calyx necrosis could be observed between E. amylovora CFBP1430 siderophore synthesis (DfoA) or uptake (FoxR receptor) mutants and the parental strain. In addition, mutant strains only weakly induced a foxR promoter-gfpmut2 reporter construct in the flowers. When analyzing the replication of the receptor mutant in apple flowers harboring an established microbiome, either naturally, in case of orchard flowers, or by pre-inoculation of semisterile greenhouse flowers, it became evident that the mutant strain had a significantly reduced replication compared to the parental strain. The results suggest that apple flowers per se are not an iron-limiting environment for E. amylovora and that DFO is an important competition factor for the pathogen in precolonized flowers. IMPORTANCE Desferrioxamine is a siderophore produced by the fire blight pathogen E. amylovora under iron-limited conditions. In the present study, no or only weak induction of an iron-regulated promoter-GFP reporter was observed on semisterile apple flowers, and siderophore synthesis or uptake (receptor) mutants exhibited colonization of the flower and necrosis induction at parental levels. Reduced replication of the receptor mutant was observed when the flowers were precolonized by microorganisms. The results indicate that apple flowers are an iron-limited environment for E. amylovora only if precolonization with microorganisms leads to iron competition. This is an important insight for the timing of biocontrol treatments.

Identification of novel virulence factors in Erwinia amylovora through temporal transcriptomic analysis of infected apple flowers under field conditions.

The enterobacterial pathogen Erwinia amylovora uses multiple virulence-associated traits to cause fire blight, a devastating disease of apple and pear trees. Many virulence-associated phenotypes have been studied that are critical for virulence and pathogenicity. Despite the in vitro testing that has revealed how these systems are transcriptionally regulated, information on when and where in infected tissues these genes are being expressed is lacking. Here, we used a high-throughput sequencing approach to characterize the transcriptome of E. amylovora during disease progression on apple flowers under field infection conditions. We report that type III secretion system genes and flagellar genes are strongly co-expressed. Likewise, genes involved in biosynthesis of the exopolysaccharide amylovoran and sorbitol utilization had similar expression patterns. We further identified a group of 16 genes whose expression is increased and maintained at high levels throughout disease progression across time and tissues. We chose five of these genes for mutational analysis and observed that deletion mutants lacking these genes all display reduced symptom development on apple shoots. Furthermore, these induced genes were over-represented for genes involved in sulphur metabolism and cycling, suggesting the possibility of an important role for maintenance of oxidative homeostasis during apple flower infection.

Early colonization of Protea flowers enable dominance of competitively weak saprobic fungi in seed cones, benefitting their hosts.

Sporothrix and Knoxdaviesia fungi use pollinators to colonize Protea flowers at anthesis. These saprobes remain dominant in the nutrient-rich, fire-retardant Protea seed-cones (infructescences) for at least a year after flowering. We tested the hypothesis that they competitively exclude potentially detrimental fungi from infructescences during this time. We compared seed set and longevity of infructescences containing Sporothrix and Knoxdaviesia vs. those that contain 'contaminant' saprobes. Hereafter we evaluated their competitive abilities against the 'contaminant' saprobes. Infructescences devoid of Sporothrix and Knoxdaviesia were dominated by Penicillium cf. toxicarium, Cladosporium cf. cladosporoides and Fusarium cf. anthophilum. Sporothrix and Knoxdaviesia presence did not affect seed viability, but infructescences persisted longer than those colonised by 'contaminant' fungi. The 'contaminant' species were stronger competitors than Sporothrix and Knoxdaviesia. However, Sporothrix and Knoxdaviesia could defend captured space well against 'contaminant' species. This effect was enhanced when fungal taxa grew on media prepared from their usual Protea host species, clarifying their dominance and host consistency observed in the field. Sporothrix and Knoxdaviesia from Protea are therefore weak competitors against common saprobes, especially when growing on alternative hosts, and need to colonise flowers very early (before colonization by other fungi) to dominate in this environment. They may delay seed release from infructescences longer than if these are colonised by other saprobes, increasing chances of seed release to occur after fire, when conditions are more favourable for Protea recruitment.

Wickerhamiella nakhonpathomensis f.a. sp. nov., a novel ascomycetous yeast species isolated from a mushroom and a flower in Thailand.

Strains SU22T (TBRC 14875T) and FLA11.5, representing a novel anamorphic yeast species, were respectively isolated from a fruiting body of a Coprinus species and an inflorescence of a Coffea species collected in Thailand. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions showed that the two strains differed by two nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and were identical in the ITS regions. Wickerhamiella drosophilae CBS 8459T was the most closely related species, but with 24-26 nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 24 nucleotide substitutions in the ITS regions. A phylogenetic analysis, based on the sequences of the D1/D2 domains, indicated that the two strains represented a species in the genus Wickerhamiella which was distinct from other recognized species of the genus. Therefore, the two strains were assigned as a novel species, for which we propose the name Wickerhamiella nakhonpathomensis f.a. sp. nov. The holotype is TBRC 14875T (isotype PYCC 8914T). The MycoBank number of the novel species is MB 840833.

Floral Microbes Suppress Growth of Monilinia laxa with Minimal Effects on Honey Bee Feeding.

Management of Monilinia laxa, the causal agent of brown rot blossom blight in almond (Prunus dulcis), relies heavily on the use of chemical fungicides during bloom. However, chemical fungicides can have nontarget effects on beneficial arthropods, including pollinators, and select for resistance in the pathogen of concern. Almond yield is heavily reliant on successful pollination by healthy honey bees (Apis mellifera); thus, identifying sustainable, effective, and pollinator-friendly control methods for blossom blight during bloom is desirable. Flower-inhabiting microbes could provide a natural, sustainable form of biocontrol for M. laxa, while potentially minimizing costly nontarget effects on almond pollinators and the services they provide. As pollinators are sensitive to floral microbes and their associated taste and scent cues, assessing effects of prospective biocontrol species on pollinator attraction is also necessary. Here, our objective was to isolate and identify potential biocontrol microbes from an array of agricultural and natural flowering hosts and test their efficacy in suppressing M. laxa growth in culture. Out of an initial 287 bacterial and fungal isolates identified, 56 were screened using a dual culture plate assay. Most strains reduced M. laxa growth in vitro. Ten particularly effective candidate microbes were further screened for their effect on honey bee feeding. Of the 10, nine were found to both strongly suppress M. laxa growth in culture and not reduce honey bee feeding. These promising results suggest a number of strong candidates for augmentative microbial biocontrol of brown rot blossom blight in almond with potentially minimal effects on honey bee pollination.

Elevated Temperature May Affect Nectar Microbes, Nectar Sugars, and Bumble Bee Foraging Preference.

Floral nectar, an important resource for pollinators, is inhabited by microbes such as yeasts and bacteria, which have been shown to influence pollinator preference. Dynamic and complex plant-pollinator-microbe interactions are likely to be affected by a rapidly changing climate, as each player has their own optimal growth temperatures and phenological responses to environmental triggers, such as temperature. To understand how warming due to climate change is influencing nectar microbial communities, we incubated a natural nectar microbial community at different temperatures and assessed the subsequent nectar chemistry and preference of the common eastern bumble bee, Bombus impatiens. The microbial community in floral nectar is often species-poor, and the cultured Brassica rapa nectar community was dominated by the bacterium Fructobacillus. Temperature increased the abundance of bacteria in the warmer treatment. Bumble bees preferred nectar inoculated with microbes, but only at the lower, ambient temperature. Warming therefore induced an increase in bacterial abundance which altered nectar sugars and led to significant differences in pollinator preference.

Comparative fungal diversity and dynamics in plant compartments at different developmental stages under root-zone restricted grapevines.

BACKGROUND: The root-zone restriction cultivation technique is used to achieve superior fruit quality at the cost of limited vegetative and enhanced reproductive development of grapevines. Fungal interactions and diversity in grapevines are well established; however, our knowledge about fungal diversity under the root-zone restriction technique is still unexplored. To provide insights into the role of mycobiota in the regulation of growth and fruit quality of grapevine under root-zone restriction, DNA from rhizosphere and plant compartments, including white roots (new roots), leaves, flowers, and berries of root-zone restricted (treatment) and conventionally grown plants (control), was extracted at three growth stages (full bloom, veraison, and maturity). RESULTS: Diversity analysis based on the ITS1 region was performed using QIIME2. We observed that the root-zone restriction technique primarily affected the fungal communities of the soil and plant compartments at different growth stages. Interestingly, Fusarium, Ilyonectria, Cladosporium and Aspergillus spp observed in the rhizosphere overlapped with the phyllosphere at all phenological stages, having distinctive abundance in grapevine habitats. Peak richness and diversity were observed in the rhizosphere at the full bloom stage of control plants, white roots at the veraison stage of treatment, leaves at the maturity stage of treatment, flowers at the full bloom stage and berries at the veraison stage of control plants. Except for white roots, the diversity of soil and plant compartments of treated plants tended to increase until maturity. At the maturity stage of the treated and control plants, the abundance of Aspergillus spp. was 25.99 and 29.48%, respectively. Moreover, the total soluble sugar content of berries was 19.03 obrix and 16 obrix in treated and control plants, respectively, at the maturity stage. CONCLUSIONS: This is the first elucidative study targeting the fungal diversity of conventional and root-restricted cultivation techniques in a single vineyard. Species richness and diversity are affected by stressful cultivation known as root zone restriction. There is an association between the abundance of Aspergillus spp. and fruit quality because despite causing stress to the grapevine, superior quality of fruit is retrieved in root-zone restricted plants.

Antagonistic Activities of Endophytic Fungi Isolated from Eleutherine palmifolia Flower.

<b>Background and Objective:</b> Endophytic fungi live in plant tissue and show no symptoms of disease in their host plants. It is known that endophytes as biological agents, can control plant diseases. In this study, isolated endophytic fungi from healthy Dayak Onion flowers were used as biocontrol agents in the control of the pathogenic fungi <i>Fusarium </i>spp. that causes molar disease in shallot plants. <b>Materials and Methods:</b> This study identifies the type of endophytic fungi molecularly isolated from Dayak onion flowers and determine the antagonistic effect of the endophytic extract against <i>Fusarium </i>spp. screening for endophytic fungi as antagonizing agents is carried out using the poisoned food method. <b>Results:</b> The results showed that two endophytic fungi isolates were obtained from healthy Dayak onion flowers, namely, EnI which was identified with the primers ITS1 and ITS4 as <i>Fusarium solani</i> and EnK as <i>Neoscytalidium </i>sp. <i>Fusarium</i> wilt caused by pathogenic fungi was identified as <i>Fusarium oxysporum</i>. The inhibitory percentage of EnI extract against the pathogenic fungus <i>Fusarium oxysporum</i> was 71.09% (high inhibition percentage) and the inhibition percentage of EnK was 38.54% (low inhibition percentage). <b>Conclusion:</b> Based on the results of this study, recommend using the endophytic fungus EnI extracts (<i>Fusarium solani </i>), extracted from Dayak onion flowers to control the pathogen <i>Fusarium oxysporum</i>.