Endophytic Fungi Residing within Cornus florida L. in Mid-Tennessee: Phylogenetic Diversity, Enzymatic Properties, and Potential Role in Plant Health.

Endophytic fungi that reside internally in healthy, asymptomatic plants often benefit their hosts by promoting plant growth and/or providing plant protection against abiotic and biotic stresses. However, only a small fraction of the estimated 1.5 million fungal endophytes have been identified. In this study, a total of 369 isolates of fungal endophytes in 59 distinct taxa were isolated from stem samples of Cornus florida (flowering dogwood). All isolates belonged to species of phyla Ascomycota and Basidiomycota distributed across five orders and 11 genera. Isolates belonging to the same family clustered together in a phylogenetic tree generated from a cluster analysis using MEGA 7 software. Diversity indices of the fungi revealed a rich and diverse community that included several species associated with leaf spots, blight, cankers, and/or dieback diseases. Pathogenicity tests confirmed 16 fungal endophytes as C. florida pathogens, including some well-known destructive pathogens Botryosphaera dothidea, Colletotrichum acutatum, and C. gleosporoides. Isolates of the fungal endophytes possess the capacity to produce extracellular hydrolytic enzymes (cellulase, amylase, pectinase, laccase, chitinase, and protease) that are known to function in tissue penetration, plant colonization, nutrient acquisition, and disease suppression in both plant pathogens and endophytes These results support the interchangeable pathogenic-endophytic roles for some taxa.

Data on plant defense enzyme activity associated with three endophytes against Cornus florida Erysiphe pulchra powdery mildew.

Three bacteria endophytes that colonize flowering dogwood (Cornus florida) suppressed Erysiphe pulchra powdery mildew disease severity. The three bacteria identified as Stenotrophomonas sp. (B17A), Serratia marcescens (B17B), and Bacillus thuringiensis (IMC8) were assessed for plant defense enzymes associated with plant protection. Detached leaves inoculated with powdery mildew were spray treated with the selected bacterial isolates and incubated for 15 h, 26 h, 48 h and 72 h and then analyzed for activation of defense enzymes and Pathogenesis related (PR) proteins associated with induced systemic resistance (ISR) as a potential mode of action against powdery mildew. At each time point post treatment with the bacteria, leaf tissue was ground in liquid nitrogen and stored at -70°C for biochemical assay of enzyme activity. This data set presents the activation of enzyme activity for peroxidase (PO), polyphenol oxidase (PPO) and ß -1,3-glucanase at 15 h, 26 h, 48 h and 72 h post treatment with bacteria as indicated by a change in absorbance min -1 mg-1 per gram fresh weight of leaves. The gene expression of the corresponding pathogenesis related (PR) protein for each bacterial treatment compared to the control was also analyzed using Real time PCR and five primers targeting PR1, PR2, and PR5. While changes for PO, PPO, and ß -1,3-glucanase enzyme activities were observed at different time points post treatment with all three bacteria, expression of PR protein was detected for PR1, but it was negligible for PR2, and PR5.

Efficient fluorescence-based localization technique for real-time tracking endophytes route in host-plants colonization.

Bacterial isolates that enhance plant growth and suppress plant pathogens growth are essential tools for reducing pesticide applications in plant production systems. The objectives of this study were to develop a reliable fluorescence-based technique for labeling bacterial isolates selected as biological control agents (BCAs) to allow their direct tracking in the host-plant interactions, understand the BCA localization within their host plants, and the route of plant colonization. Objectives were achieved by developing competent BCAs transformed with two plasmids, pBSU101 and pANIC-10A, containing reporter genes eGFP and pporRFP, respectively. Our results revealed that the plasmid-mediated transformation efficiencies of antibiotic-resistant competent BCAs identified as PSL, IMC8, and PS were up 84%. Fluorescent BCA-tagged reporter genes were associated with roots and hypocotyls but not with leaves or stems and were confirmed by fluoresence microscopy and PCR analyses in colonized Arabidopsis and sorghum. This fluorescence-based technique's high resolution and reproducibility make it a platform-independent system that allows tracking of BCAs spatially within plant tissues, enabling assessment of the movement and niches of BCAs within colonized plants. Steps for producing and transforming competent fluorescent BCAs, as well as the inoculation of plants with transformed BCAs, localization, and confirmation of fluorescent BCAs through fluorescence imaging and PCR, are provided in this manuscript. This study features host-plant interactions and subsequently biological and physiological mechanisms implicated in these interactions. The maximum time to complete all the steps of this protocol is approximately 3 months.

Sources of Resistance to Common Bacterial Blight and Charcoal Rot Disease for the Production of Mesoamerican Common Beans in the Southern United States.

The gene pool of Mesoamerican common beans (Phaseolus vulgaris L.) includes genotypes in the small-to-medium-size seeded dry beans, as well as some snap beans from hotter environments adapted to the Southeastern United States. However, the warm and humid climate of the Southeastern United States is conducive to diseases such as Common Bacterial Blight (CBB) and Charcoal Rot (CR). The pathogens for these two diseases can survive long periods in infested soil or on seeds and are difficult to control through pesticides. Hence, field-level resistance would be the best management strategy for these diseases. The goals of this study were (1) to evaluate field-level resistance from the various commercial classes and subgroups represented in the Mesoamerican gene pool as sources for breeding beans for the region and (2) to evaluate genome-wide marker × trait associations (GWAS) using genetic markers for the genotypes. A total of 300 genotypes from the Mesoamerican Diversity Panel (MDP) were evaluated for CBB and CR in field experiments for three years. CBB resistance was also tested with a field isolate in controlled greenhouse conditions. The analysis of variance revealed the presence of variability in the MDP for the evaluated traits. We also identified adapted common bean genotypes that could be used directly in Southeastern production or that could be good parents in breeding programs for CBB and CR resistance. The GWAS detected 14 significant Single-Nucleotide Polymorphism (SNP) markers associated with CBB resistance distributed on five chromosomes, namely Pv02, Pv04, Pv08, Pv10, and Pv11, but no loci for resistance to CR. A total of 89 candidate genes were identified in close vicinity (±100 kb) to the significant CBB markers, some of which could be directly or indirectly involved in plant defense to diseases. These results provide a basis to further understand the complex inheritance of CBB resistance in Mesoamerican common beans and show that this biotic stress is unrelated to CR resistance, which was evident during a drought period. Genotypes with good yield potential for the Southeastern U.S. growing conditions were found with resistant to infection by the two diseases, as well as adaptation to the hot and humid conditions punctuated by droughts found in this region.

Perpetuation of Powdery Mildew Infection and Identification of Erysiphe australiana as the Crape Myrtle Pathogen in Mid-Tennessee.

The fungus Erysiphe lagerstroemiae is commonly known as the powdery mildew pathogen in crape myrtle (Lagerstroemiae indica) in the United States, and Erysiphe australiana is the powdery mildew pathogen reported in Japan, China, and Australia. The teleomorph often used to identify powdery mildew fungi rarely develops in crape myrtle, and in our observations, ascocarps never formed. Our study showed that the crape myrtle pathogen overwintered as mycelia on dormant buds. The internal transcribed spacer (ITS) regions of rDNA and the intervening 5.8S rRNA gene were amplified using standard polymerase chain reaction (PCR) protocols and the universal primer pairs ITS1 and ITS4. PCR products were analyzed by electrophoresis in a 1.5% agarose gel and sequenced, and the ITS PCR product was 666 bp from ITS1/ITS4 and 704 bp from ITS1-F/ITS4. BLAST analysis of the sequence of the PCR products showed identical similarity with E. australiana reported in Japan, China, and Australia. Comparison of ITS sequences with information in the GenBank on other powdery mildew fungi showed a closest alignment (93% similarity) to Erysiphe juglandis that infects walnut. Specific primers for E. australiana were developed and evaluated for use as diagnostic tools. Out of 12 specific primer pairs evaluated, four primer pairs and four double primer pairs were highly specific to E. australiana and did not amplify Erysiphe pulchra of dogwood, Erysiphe syringae of common lilac, Erysiphe circinata of maple, or Phyllactinia guttata of oak. The E. australiana-specific primers amplified 16 samples of crape myrtle powdery mildew collected from diverse locations in mid-Tennessee. These results clearly showed that the crape myrtle powdery mildew in mid-Tennessee was caused by E. australiana. Specific primers reported in this article provide a diagnostic tool and may be used to confirm the identity of crape myrtle powdery mildew pathogen in other areas in the United States and wherever the disease occurs.

Winter Survival and Source of Primary Inoculum of Powdery Mildew of Dogwood in Tennessee.

A 3-year study (1996-1998) on the epidemiology of dogwood powdery mildew showed that Microsphaera pulchra is the primary powdery mildew pathogen of dogwoods (Cornus spp.) in mid-Tennessee, and the occurrence of Phyllactinia guttata is insignificant. Cleistothecia harvested from leaf debris in spring contained viable asci and ascospores and produced powdery mildew infection on disease-free plants. Ascospores that were morphologically similar to those of M. pulchra were trapped on sticky slides in the vicinity of dogwoods throughout spring. Previously infected plants that did not harbor cleistothecia failed to develop signs of infection under growth-chamber and greenhouse conditions conducive to powdery mildew infections. Results from these studies indicate that cleistothecia on leaf debris rather than mycelia on dormant buds constitute the main winter survival structure and primary sources of spring inoculum in mid-Tennessee.