Boxwood phyllosphere fungal and bacterial communities and their differential responses to film-forming anti-desiccants.

BACKGROUND: Anti-desiccant is a class of agrochemicals widely used to protect plants from water stresses, rapid temperature variations, heat and sunburn, frost and freeze damages, transplant shock, and pathogen and pest attack. Although anti-desiccants are generally considered non-toxic to organisms, it is unclear whether they may impact the phyllosphere microbial communities. In this study, three film-forming anti-desiccant products, TransFilm, Vapor Gard, and Wilt-Pruf were applied to the canopy of two boxwood cultivars 'Vardar Valley' and 'Justin Brouwers' on April 13 and August 26, 2021. Shoot samples were collected from boxwood plants treated with each of the three products, as well as nontreated control on June 16, August 26 (before the second treatment), and October 18. Microbial and plant genomic DNA was isolated together and 16S rRNA gene and the extended internal transcribed spacer regions were amplified with PCR and sequenced on a Nanopore MinION platform for bacterial and fungal identification. RESULTS: Bacterial communities were more diverse than fungal communities. At the phylum level, the boxwood phyllosphere was dominated by Proteobacteria and Ascomycota; at the genus level, Methylobacterium and Shiraia were the most abundant bacteria and fungi, respectively. Among the three film-forming anti-desiccants, Vapor Gard and Wilt-Pruf had more impact than TransFilm on the microbial communities. Specifically, broader impacts were observed on fungal than bacterial community composition and structure, with most affected fungi being suppressed while bacteria promoted. CONCLUSION: This study addressed several major knowledge gaps regarding boxwood phyllosphere microbiota and the impact of anti-desiccants on plant microbiome. We identified diverse microbial communities of boxwood, a major evergreen woody crop and an iconic landscape plant. We also found differential effects of three film-forming anti-desiccants on the composition and structure of bacterial and fungal communities. These findings advanced our understanding of the associated microbiome of this landmark plant, enabling growers to fully utilize the potentials of microbiome and three anti-desiccants in improving boxwood health and productivity.

Targeting the Aspergillus flavus p2c gene through host-induced gene silencing reduces A. flavus infection and aflatoxin contamination in transgenic maize.

Aspergillus flavus is an opportunistic fungal pathogen that infects maize and produces aflatoxins. Using biocontrol or developing resistant cultivars to reduce aflatoxin contamination has only achieved limited success. Here, the A. flavus polygalacturonase gene (p2c) was targeted for suppression through host-induced gene silencing (HIGS) to reduce aflatoxin contamination in maize. An RNAi vector carrying a portion of the p2c gene was constructed and transformed into maize B104. Thirteen out of fifteen independent transformation events were confirmed to contain p2c. The T2 generation kernels containing the p2c transgene had less aflatoxin than those without the transgene in six out of eleven events we examined. Homozygous T3 transgenic kernels from four events produced significantly less aflatoxins (P ≤ 0.02) than the kernels from the null or B104 controls under field inoculation conditions. The F1 kernels from the crosses between six elite inbred lines with P2c5 and P2c13 also supported significantly less aflatoxins (P ≤ 0.02) than those from the crosses with null plants. The reduction in aflatoxin ranged from 93.7% to 30.3%. Transgenic leaf (T0 and T3) and kernel tissues (T4) were also found to have significantly higher levels of p2c gene-specific small RNAs. Further, homozygous transgenic maize kernels had significantly less fungal growth (27~40 fold) than the null control kernels 10 days after fungal inoculation in the field. The calculated suppression of p2c gene expression based on RNAseq data was 57.6% and 83.0% in P2c5 and P2c13 events, respectively. These results indicate clearly that the reduced aflatoxin production in the transgenic kernels is due to RNAi-based suppression of p2c expression, which results in reduced fungal growth and toxin production.

Characterization of Boxwood Shoot Bacterial Communities and Potential Impact from Fungicide Treatments.

Phyllosphere bacterial communities play important roles in plant fitness and growth. The objective of this study was to characterize the epiphytic and endophytic bacterial communities of boxwood shoots and determine how they may respond to commonly used fungicides. In early summer and early fall, shoot samples were collected immediately before and 1, 7, and 14 days after three fungicides containing chlorothalonil and/or propiconazole were applied to the canopy. Total genomic DNA from shoot surface washings and surface-sterilized shoot tissues was used as the template for 16S rRNA metabarcoding, and the amplicons were sequenced on a Nanopore MinION sequencer to characterize the epiphytic and endophytic communities. The bacterial communities were phylogenetically more diverse on the boxwood shoot surface than in the internal tissue, although the two communities shared 12.7% of the total 1,649 identified genera. The most abundant epiphytes were Methylobacterium and Pantoea, while Stenotrophomonas and Brevundimonas were the dominant endophytes. Fungicide treatments had strong impacts on epiphytic bacterial community structure and composition. Analysis of compositions of microbiomes with bias correction (ANCOM-BC) and analysis of variance (ANOVA)-like differential expression (ALDEx2) together identified 312 and 1,362 epiphytes changed in abundance due to fungicide treatments in early summer and early fall, respectively, and over 50% of these epiphytes were negatively impacted by fungicide. The two chlorothalonil-based contact fungicides demonstrated more marked effects than the propiconazole-based systemic fungicide. These results are foundational for exploring and utilizing the full potential of the microbiome and fungicide applications and developing a systems approach to boxwood health and production. IMPORTANCE Agrochemicals are important tools for safeguarding plants from invasive pathogens, insects, mites, and weeds. How they may affect the plant microbiome, a critical component of crop health and production, was poorly understood. Here, we used boxwood, an iconic low-maintenance landscape plant, to characterize shoot epiphytic and endophytic bacterial communities and their responses to contact and systemic fungicides. This study expanded our understanding of the above-ground microbiome in ornamental plants and is foundational for utilizing the full benefits of the microbiome in concert with different fungicide chemistries to improve boxwood health. This study also sets an example for a more thorough evaluation of these and other agrochemicals for their effects on boxwood microbiomes during production and offers an expanded systems approach that could be used with other crops for enhanced integrated pest management.

Combating an Invasive Boxwood Pathogen - Calonectria pseudonaviculata - in the United States by Shifting Production to Less-Susceptible Cultivars.

Boxwood blight (BB) caused by Calonectria pseudonaviculata (Cps), an economically devastating disease affecting everyone in the supply chain from growers to gardeners, was first officially documented in the United States in 2011. This disease has taken a heavy toll on boxwood, an iconic landscape plant and the number one evergreen nursery crop. Instead of abandoning boxwood production and switching to other evergreens, growers in the United States, informed by the latest research, have elected to combat this disease. One of the strategies employed has been to shift boxwood production from highly susceptible to less-susceptible cultivars. The objective of this study was to investigate the ongoing shift by comparing boxwood sales of 17 selected nurseries from seven states across the country in 2011, 2016, and 2021. Results revealed an additional 21.24% (from 38.58 to 60.04%) in sales of less-susceptible boxwood cultivars in 2021 compared with 2016. The less-susceptible cultivars responsible for increased boxwood sales are 'Winter Gem', 'Wintergreen', 'SB 300' (Freedom), 'SB 108' (Independence), and 'Little Missy'. The potential for long-term positive impact on sustainable boxwood production and plantings in the United States through the use of less-susceptible cultivars is discussed. This shift in boxwood choices builds crop health into new plantings of this landmark plant and sustains growth in demand for boxwood. This sets a new example of sustainable protection for a crop that is under serious pressure from an invasive pathogen.

Resistance to Aflatoxin Accumulation in Maize Mediated by Host-Induced Silencing of the Aspergillus flavus Alkaline Protease (alk) Gene.

Aspergillus flavus is a fungal pathogen that infects maize and produces aflatoxins. Host-Induced Gene Silencing (HIGS) has been shown to reduce host infection by various fungal pathogens. Here, the A. flavus alkaline protease (alk) gene was targeted for silencing through HIGS. An RNAi vector carrying a portion of the alk gene was incorporated into the B104 maize genome. Four out of eight transformation events containing the alk gene, Alk-3, Alk-4, Alk-7 and Alk-9, were self-pollinated to T4/T6 generations. At T3, the Alk-transgenic lines showed up to 87% reduction in aflatoxin accumulation under laboratory conditions. T4 transgenic Alk-3 and Alk-7 lines, and T5 and T6 Alk-4 and Alk-9 showed an average of 84% reduction in aflatoxin accumulation compared to their null controls under field inoculations (p < 0.05). F1 hybrids of three elite maize inbred lines and the transgenic lines also showed significant improvement in aflatoxin resistance (p < 0.006 to p < 0.045). Reduced A. flavus growth and levels of fungal ß-tubulin DNA were observed in transgenic kernels during in vitro inoculation. Alk-4 transgenic leaf and immature kernel tissues also contained about 1000-fold higher levels of alk-specific small RNAs compared to null controls, indicating that the enhanced aflatoxin resistance in the transgenic maize kernels is due to suppression of A. flavus infection through HIGS of alk gene.

Host Induced Gene Silencing Targeting Aspergillus flavus aflM Reduced Aflatoxin Contamination in Transgenic Maize Under Field Conditions.

Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. This pathogen can pose serious health concerns and cause severe economic losses due to the Food and Drug Administration (FDA) regulations on permissible levels of aflatoxins in food and feed. Although biocontrol has yielded some successes in managing aflatoxin contamination, enhancing crop resistance is still the preferred choice of management for long-term sustainability. Hence, host induced gene silencing (HIGS) strategy was explored in this study. The A. flavus gene aflM encoding versicolorin dehydrogenase, a key enzyme involved in the aflatoxin biosynthetic pathway, was selected as a possible target for suppression through HIGS. An RNAi vector containing a portion of the aflM gene was constructed and introduced into immature B104 maize zygotic embryos through Agrobacterium transformation. PCR analysis of the genomic DNA from T0 leaf tissue confirmed the presence of the transgene in six out of the seven events. The seeds from the lines that showed reduced aflatoxin production in laboratory aflatoxin kernel screening assay (KSA) have been increased from T1 to T4 generation in the past four years. Changes in aflatoxin resistance in these transgenic kernels have been evaluated under both field and laboratory conditions. The T2 generation kernels containing the transgene from two events out of four examined had less aflatoxin (P ≤ 0.01 and P ≤ 0.08) than those without the transgene. Field-inoculated homozygous T3 and T4 transgenic kernels also revealed lower levels of aflatoxins (P ≤ 0.04) than kernels from the null (segregated non-transgenic samples) or B104 controls. A similar result was observed when the harvested T3 and T4 homozygous transgenic kernels were evaluated under KSA conditions without inoculation (P ≤ 0.003-0.05). These two events were crossed with LH195, LH197, LH210, and PHW79 elite breeding lines and the resulting crosses supported less aflatoxin (P ≤ 0.02) than the crosses made with non-transgenic lines. In addition, significantly higher levels of aflM gene-specific small RNAs were detected in the transgenic leaf and kernel tissues, indicating that the enhanced aflatoxin resistance in the homozygous transgenic kernels is likely due to suppression of aflM expression through HIGS.