Fusarium oxysporum as an Opportunistic Fungal Pathogen on Zinnia hybrida Plants Grown on board the International Space Station.

A plant production system called Veggie was launched to the International Space Station (ISS) in 2014. In late 2015, during the growth of Zinnia hybrida cv. 'Profusion' in the Veggie hardware, plants developed chlorosis, leaf curling, fungal growth that damaged leaves and stems, and eventually necrosis. The development of symptoms was correlated to reduced air flow leading to a significant buildup of water enveloping the leaves and stems in microgravity. Symptomatic tissues were returned to Earth on 18 May 2016 and were immediately processed to determine the primary causal agent of the disease. The presumptive pathogen was identified as Fusarium oxysporum by morphological features of microconidia and conidiophores on symptomatic tissues; that is, by epifluorescent microscopy (EFM), scanning electron microscopy (SEM), metabolic microarrays, and ITS sequencing. Both EFM and SEM imaging of infected tissues showed that germinating conidia were capable of stomatal penetration and thus acted as the primary method for infecting host tissues. A series of ground-based pathogenicity assays were conducted with healthy Z. hybrida plants that were exposed to reduced-airflow and high-water stress (i.e., encased in sealed bags) or were kept in an unstressed configuration. Koch's postulates were successfully completed with Z. hybrida plants in the lab, but symptoms only matched ISS-flown symptomatic tissues when the plants were stressed with high-water exposure. Unstressed plants grown under similar lab conditions failed to develop the symptoms observed with plants on board the ISS. The overall results of the pathogenicity tests imply that F. oxysporum acted as an opportunistic pathogen on severely high-water stressed plants. The source of the opportunistic pathogen is not known, but virulent strains of F. oxysporum were not recovered from unused materials in the Veggie plant pillow growth units assayed after the flight.

Transcriptome Profiling of Buffalograss Challenged with the Leaf Spot Pathogen Curvularia inaequalis.

Buffalograss (Bouteloua dactyloides) is a low maintenance U. S. native turfgrass species with exceptional drought, heat, and cold tolerance. Leaf spot caused by Curvularia inaequalis negatively impacts buffalograss visual quality. Two leaf spot susceptible and two resistant buffalograss lines were challenged with C. inaequalis. Samples were collected from treated and untreated leaves when susceptible lines showed symptoms. Transcriptome sequencing was done and differentially expressed genes were identified. Approximately 27 million raw sequencing reads were produced per sample. More than 86% of the sequencing reads mapped to an existing buffalograss reference transcriptome. De novo assembly of unmapped reads was merged with the existing reference to produce a more complete transcriptome. There were 461 differentially expressed transcripts between the resistant and susceptible lines when challenged with the pathogen and 1552 in its absence. Previously characterized defense-related genes were identified among the differentially expressed transcripts. Twenty one resistant line transcripts were similar to genes regulating pattern triggered immunity and 20 transcripts were similar to genes regulating effector triggered immunity. There were also nine up-regulated transcripts in resistance lines which showed potential to initiate systemic acquired resistance (SAR) and three transcripts encoding pathogenesis-related proteins which are downstream products of SAR. This is the first study characterizing changes in the buffalograss transcriptome when challenged with C. inaequalis.

Porocercospora seminalis gen. et comb. nov., the causal organism of buffalograss false smut.

False smut caused by Cercospora seminalis is an important disease of buffalograss (Buchloë dactyloides) affecting seed production. The pathogen prevents normal caryopsis development and causes considerable yield loss and reduced seed germination. The current taxonomic placement of the false-smut causal pathogen in the genus Cercospora is incorrect based on its morphological characteristics and DNA phylogeny. In the present study the phylogenetic position of C. seminalis is clarified based on DNA sequence analysis of three loci namely the internal transcribed spacer (ITS) region, partial nuclear ribosomal large subunit (LSU) and partial sequences of the RNA polymerase II second largest subunit (RPB2). A collection of C. seminalis isolates was made from buffalograss sites near Lincoln, Nebraska. DNA sequence data indicated that Cercospora seminalis is phylogenetically close to but distinct from species of Bipolaris and Curvularia (Pleosporaceae, Pleosporales). Cercospora seminalis morphologically had unique characteristics, namely densely aggregated and repeatedly branched conidiophores arising from a brown stroma, monotretic conidiogenous cells with inconspicuous loci, and scolecosporous conidia with distosepta, and thickened, darkened hila. Porocercospora is introduced as a new genus to accommodate the buffalograss false-smut pathogen.

Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses.

A rapid identification assay for Waitea circinata (anamorph: Rhizoctonia spp.) varieties zeae and circinata causing patch diseases on turfgrasses was developed based on the universally primed PCR (UP-PCR) products cross-blot hybridization. Tester isolates belonging to the two varieties of W. circinata were amplified with a single UP primer L21, which generated multiple DNA fragments for each variety. Probes were prepared with UP-PCR products of each tester isolate by labeling with digoxigenin. Fieldcollected W. circinata isolates and representative isolates of different R. solani anastomosis groups (AG) and AG subgroups were amplified with L21, immobilized on nylon membrane and cross hybridized with the two probes. Isolates within a W. circinata variety cross-hybridized strongly, while non-homologous isolates did not cross-hybridize or did so weakly. Closely related W. circinata varieties zeae and circinata were clearly distinguished with this assay. Sequence-characterized amplified region (SCAR) markers also were developed from UP-PCR products to identify isolates of Thanatephorus cucumeris (anamorph: R. solani) AG 1-IB and AG 2-2IIIB. These two AGs are commonly isolated from diseased, cool-season turfgrasses. The specific SCAR markers that were developed could differentiate isolates of AG 1-IB or AG 2-2IIIB groups. These SCAR markers did not amplify a product from genomic DNA of nontarget isolates of Rhizoctonia. The specificities and sensitivities of the SCAR primers were tested on total DNA extracted from several field-grown, cool-season turf species having severe brown-patch symptoms. First, the leaf samples from diseased turf species were tested for the anastomosis groups of the causal pathogen, and thereafter the total DNA was amplified with the specific primers. The specific primers were sensitive and unique enough to produce a band from total DNA of diseased turfgrasses infected with either AG 1-IB or AG 2-2IIIB.

Vertical migration of Haemonchus contortus infective larvae on Cynodon dactylon and Paspalum notatum pastures in response to climatic conditions.

Observations were made on vertical migration patterns of Haemonchus contortus infective larvae on Cynodon dactylon (bermudagrass) and Paspalum notatum (bahiagrass) pastures under summer climatic conditions typical of East Texas. Ten thousand H. contortus infective larvae (L3) were introduced to 100 cm(2) subplots of each pasture species within a plot area of 1m(2). Subplots were inoculated with larvae by applying them in an aqueous medium to the soil or mat beneath the vegetation. Herbage from the inoculated areas was harvested on 5 sampling days over a span of 21 days. L3 recoveries were observed and recorded each day on four herbage strata viz. 0-5, 5-10, 10-20 and >20 cm from ground level. The log transformed larval recovery data were analyzed for effect of day, stratum, and day x stratum interaction for each grass species during two separate experimental periods. Precipitation, relative humidity and temperature during the study were subjected to correlation and multiple regression analyses with the larval counts. Significant (Por=0.93) between rainfall and total average daily larval counts was apparent. The multiple regression analysis did not show significant results for any of the climatic factors tested. This study showed that the H. contortus infective larvae can survive beyond 21 days in the soil and infest pasture grasses when the climatic conditions are favorable. Avoiding use of H. contortus contaminated pasturelands in summer at the onset of rainfall following a dry spell may effectively reduce nematode loads in susceptible farm animals. Additional studies should focus on factors affecting long term L3 survivability, migrational pattern on these and other plant species and the relationship between climatic factors and larval migration patterns throughout the year. Total larval recovery of H. contortus in this study was greater in bahiagrass than bermudagrass. While the design of this study did not allow for testing one pasture species against another, studies with potted plants would allow for some valid comparisons. Soil characteristics may also play a role in L3 survival and subsequent migration.