Bacterial Endophyte Community Dynamics in Apple (Malus domestica Borkh.) Germplasm and Their Evaluation for Scab Management Strategies

The large genetic evolution due to the sexual reproduction-mediated gene assortments and propensities has made Venturia inaequalis (causing apple scab) unique with respect to its management strategies. The resistance in apple germplasm against the scab, being controlled for by more than fifteen genes, has limited gene alteration-based investigations. Therefore, a biological approach of bacterial endophyte community dynamics was envisioned across the apple germplasm in context to the fungistatic behavior against V. inaequalis. A total of 155 colonies of bacterial endophytes were isolated from various plant parts of the apple, comprising 19 varieties, and after screening for antifungal behavior followed by morphological, ARDRA, and sequence analysis, a total of 71 isolates were selected for this study. The alpha diversity indices were seen to fluctuate greatly among the isolation samples in context to microflora with antifungal behavior. As all the isolates were screened for the presence of various metabolites and some relevant genes that directly or indirectly influence the fungistatic behavior of the isolated microflora, a huge variation among the isolated microflora was observed. The outstanding isolates showing highest percentage growth inhibition of V. inaequalis were exploited to raise a bio-formulation, which was tested against the scab prevalence in eight apple varieties under controlled growth conditions. The formulation at all the concentrations caused considerable reductions in both the disease severity and disease incidence in all the tested apple varieties. Red Delicious being most important cultivar of the northwestern Himalayas was further investigated for its biochemical behavior in formulation and the investigation revealed different levels of enzyme production, chlorophyll, and sugars against the non-inoculated control.

Differential Tolerance to Calonectria pseudonaviculata of English Boxwood Plants Associated with the Complexity of Culturable Fungal and Bacterial Endophyte Communities

Isolated boxwood endophytes have been demonstrated to effectively protect boxwood plants from infection by Calonectria pseudonaviculata (Cps). However, the roles of endophytes as communities in plant defense are not clear. Here, we demonstrated differential tolerance to Cps of English boxwood (Buxus sempervirens ‘Suffruticosa’), an iconic landscape plant and generally regarded as highly susceptible, and its link to endophyte complexity. Fifteen boxwood twig samples were collected in triplicates from three historic gardens—Colonial Williamsburg, George Washington’s Mount Vernon and River Farm, and Virginia Tech’s research farm in Virginia Beach in the summer and fall of 2019. A portion of individual samples was inoculated with Cps under controlled conditions. Significant differences in disease severity were observed among samples but not between the two seasons. Examining the endophyte cultures of the summer samples revealed that bacterial and fungal abundance was negatively and positively correlated with the disease severity. Nanopore metagenomics analysis on genomic DNA of the tolerant and susceptible group representatives confirmed the associations. Specifically, tolerant English boxwood plants had an endophyte community dominated by Bacilli and Betaproteobacteria, while susceptible ones had a distinct endophyte community dominated by Gammaproteobacteria, Actinobacteria, and diverse fungi. These findings may lead to boxwood health management innovations—devising and utilizing cultural practices to manipulate and increase the abundance and performance of beneficial endophytes for enhanced boxwood resistance to Cps.

Apple endophyte community in relation to location, scion and rootstock genotypes and susceptibility to European canker

ABSTRACT European apple canker, caused by Neonectria ditissima, is a severe disease of apple. Achieving effective control is difficult with the currently available pesticides. Specific apple endophytes associated with cultivars may partially contribute to the cultivar response to the pathogen and thus could be used for disease management. We sought to determine whether the overall endophyte community differed among cultivars differing in their susceptibility to N. ditissima and to identify specific microbial groups associated with the susceptibility. Using Illumina MiSeq meta-barcoding, we profiled apple tree endophytes in 16 scion–rootstock combinations at two locations and quantified the relative contribution of scion, rootstock and location to the observed variability in the endophyte communities. Endophyte diversity was primarily affected by the orchard location (accounting for 29.4% and 85.9% of the total variation in the PC1 for bacteria and fungi, respectively), followed by the scion genotype (24.3% and 19.5% of PC2), whereas rootstock effects were small (<3% of PC1 and PC2). There were significant differences in the endophyte community between canker-resistant and -susceptible cultivars. Several bacterial and fungal endophyte groups had different relative abundance between susceptible and resistant cultivars. These endophyte groups included putative pathogen antagonists as well as plant pathogens. Their possible ecological roles in the N. ditissima pathosystem are discussed.

Antibiotic Resistance Gene Transformation and Ultrastructural Alterations of Lettuce (Lactuca sativa L.) Resulting from Sulfadiazine Accumulation in Culture Solution

The research herein explored the possible mechanism of toxicity of the antibiotic sulfadiazine (SD) and the related antibiotic resistance gene transformation in lettuce by systematically investigating its growth responses, ultrastructural changes, and antibiotic resistance gene transformation via solution culture experiments. The results showed that SD mainly accumulated in the roots of lettuce at concentrations ranging from 6.48 to 120.87 μg/kg, which were significantly higher than those in leaves (3.90 to 16.74 μg/kg). Lower concentrations of SD (0.5 and 2.0 mg/L) in the culture nutrient solution exerted little effect on lettuce growth, while at SD concentrations higher than 10 mg/L, the growth of lettuce was significantly inhibited, manifesting as shorter root length and lower dry matter yield of whole lettuce plants. Compared with that for the control group, the absolute abundance of bacteria in the root endophyte, rhizosphere, and phyllosphere communities under different concentrations of SD treatment decreased significantly. sul1 and sul2 mainly accumulated in the root endophyte community, at levels significantly higher than those in the leaf endophyte community. Studies of electrolyte leakage and ultrastructural characteristics of root and leaf cells indicated that lettuce grown in culture solutions with high SD concentrations suffered severe damage and disintegration of the cell walls of organs, especially chloroplasts, in leaves. Furthermore, the possible mechanism of SD toxicity in lettuce was confirmed to start with the roots, followed by a free flow of SD into the leaves to destroy the chloroplasts in the leaf cells, which ultimately reduced photosynthesis and decreased plant growth. Studies have shown that antibiotic residues have negative effects on the growth of lettuce and highlight a potential risk of the development and spread of antibiotic resistance in vegetable endophyte systems.

Why Plants Harbor Complex Endophytic Fungal Communities: Insights From Perennial Bunchgrass Stipagrostis sabulicola in the Namib Sand Sea

All perennial plants harbor diverse endophytic fungal communities, but why they tolerate these complex asymptomatic symbioses is unknown. Using a multi-pronged approach, we conclusively found that a dryland grass supports endophyte communities comprised predominantly of latent saprophytes that can enhance localized nutrient recycling after senescence. A perennial bunchgrass, Stipagrostis sabulicola, which persists along a gradient of extreme abiotic stress in the hyper-arid Namib Sand Sea, was the focal point of our study. Living tillers yielded 20 fungal endophyte taxa, 80% of which decomposed host litter during a 28-day laboratory decomposition assay. During a 6-month field experiment, tillers with endophytes decomposed twice as fast as sterilized tillers, consistent with the laboratory assay. Furthermore, profiling the community active during decomposition using next-generation sequencing revealed that 59–70% of the S. sabulicola endophyte community is comprised of latent saprophytes, and these dual-niche fungi still constitute a large proportion (58–62%) of the litter community more than a year after senescence. This study provides multiple lines of evidence that the fungal communities that initiate decomposition of standing litter develop in living plants, thus providing a plausible explanation for why plants harbor complex endophyte communities. Using frequent overnight non-rainfall moisture events (fog, dew, high humidity), these latent saprophytes can initiate decomposition of standing litter immediately after tiller senescence, thus maximizing the likelihood that plant-bound nutrients are recycled in situ and contribute to the nutrient island effect that is prevalent in drylands.

Foliar endophytes in trees varying greatly in age

Temple trees, including the gymnosperm Platycladus orientalis and the angiosperm Styphnolobium japonicum, have been planted in China for thousands of years. Tree age thus varies widely from young to ancient trees. Foliar endophytes of P. orientalis and S. japonicum were surveyed in this exploratory study that was based on isolation into culture and sequencing of fungi from trees varying in age from 10 to 5000 years (P. orientalis) and from 10 to 1700 years (S. japonicum). Sequenced endophytes of P. orientalis and S. japonicum belonged to 24 and 16 fungal genera, respectively. Principal components analysis showed that 14 components were necessary to explain 90% of the variance in endophyte community structure in P. orientalis. In S. japonicum eight components were needed for 90%. It is against that backdrop of complex etiology in community structuring, that the relative frequencies (abundances) of 17 of the 24 endophytes from P. orientalis and 9 of the 16 from S. japonicum were significantly correlated with tree age. There were two major trends. Abundant fungal genera [Fusarium + Alternaria = 74.57% (P. orientalis) and 81.24% (S. japonicum)] tended to decline linearly with tree age. Most of the rare fungal genera, in contrast, increased in relative abundance linearly with tree age. Diversity (H′) and richness (Margalef) of endophyte communities in foliage thus increased as the trees aged. Relative abundances of pathogenic endophytes, or latent pathogens, (Pestalotiopsis funerea and Amyloporia subxantha in P. orientalis; Collectotrichum gloeosporioides and Botryosphaeria dothidea in S. japonicum) also increased linearly as the trees aged.Since leaf age does not vary with tree age in the deciduous S. japonicum, nor in the evergreen P. orientalis, ‘tree age’ currently lacks a mechanistic explanation for its apparent importance among common foliar endophytes.

Survival of the fittest: How the rice microbial community forces Sarocladium oryzae into pathogenicity

ABSTRACT The fungus Sarocladium oryzae (Sawada) causes rice sheath rot and produces the phytotoxins cerulenin and helvolic acid. Both toxins show antimicrobial activity but only helvolic acid production in the rice sheath correlates with virulence. S. oryzae isolates that differ in their toxin production were used to study their interaction with the rice culturable bacterial endophyte community. The diversity and community structure was defined in the edge of sheath rot lesions, followed by a null model-based co-occurrence analysis to discover pairwise interactions. Non-random pairs were co-cultured to study the nature of the interactions and the role of the toxins herein. Compared to healthy sheaths, endophyte diversity strongly increased when infected with the least virulent S. oryzae isolates producing low amounts of toxins. Virulent S. oryzae isolates did not affect diversity but caused strong shifts in species composition. The endophyte community of healthy rice plants was dominated by B. cereus. This bacterium was enriched in lesions produced by low-virulent S. oryzae isolates and caused hyphal lysis. Contrarily, helvolic acid producers eliminated this bacterium from the sheath endosphere. We conclude that S. oryzae needs to produce antibiotics to defend itself against antagonistic rice endophytes to successfully colonize and infect the rice sheath.