Rhizoctonia Resistance Is Negatively Correlated to Early Root Growth Rate in Synthetic Hexaploid Wheat Derivatives.

Resistance to the soilborne fungal pathogen Rhizoctonia solani AG-8 is desirable in adapted wheat and barley but remains an elusive trait for prebreeders and breeders. In a previous study, we observed that emergence and root growth was faster in the Rhizoctonia-susceptible 'Scarlet' than in its resistant counterpart, 'Scarlet-Rz1'. The objective of the current study was to quantify early root growth rate and total root length in resistant and susceptible synthetic hexaploid wheat lines, including parental lines and 22 recombinant inbred lines derived crosses between parental lines. In Petri dish assays, the susceptible lines displayed a faster rate of root growth during the first 40 h of root emergence compared with resistant lines. This growth differential was observed in 14-day and 48-h greenhouse assays, in which the total root length of susceptible parental lines was significantly (P < 0.05) greater than that of resistant parental lines. However, the resistant lines sustained less root loss compared with susceptible lines when R. solani AG-8 was present in the soil. Early root growth rate and total root length were not correlated to freezing tolerance in a set of wheat cultivars selected for cold tolerance. The findings indicated that early root growth was negatively correlated to R. solani AG-8 damage in resistant synthetic wheat lines developed for the Pacific Northwest, United States, and suggested that the dynamics of root emergence affect resistance to this soilborne pathogen.

Proteome-Wide Response of Dormant Caryopses of the Weed, Avena fatua, After Colonization by a Seed-Decay Isolate of Fusarium avenaceum.

Promoting seed decay is an ecological approach to reducing weed persistence in the soil seedbank. Previous work demonstrated that Fusarium avenaceum F.a.1 decays dormant Avena fatua (wild oat) caryopses and induces several defense enzyme activities in vitro. The objectives of this study were to obtain a global perspective of proteins expressed after F.a.1-caryopsis colonization by conducting proteomic evaluations on (i) leachates, soluble extrinsic (seed-surface) proteins released upon washing caryopses in buffer and (ii) proteins extracted from whole caryopses; interactions with aluminum (Al) were also evaluated in the latter study because soil acidification and associated metal toxicity are growing problems. Of the 119 leachate proteins classified as defense/stress, 80 were induced or repressed. Defense/stress proteins were far more abundant in A. fatua (35%) than in F.a.1 (12%). Avena defense/stress proteins were also the most highly regulated category, with 30% induced and 35% repressed by F.a.1. Antifungal proteins represented 36% of Avena defense proteins and were the most highly regulated, with 36% induced and 37% repressed by F.a.1. These results implicate selective regulation of Avena defense proteins by F.a.1. Fusarium proteins were also highly abundant in the leachates, with 10% related to pathogenicity, 45% of which were associated with host cell wall degradation. In whole caryopsis extracts, fungal colonization generally resulted in induction of a similar set of Avena proteins in the presence and absence of Al. Results advance the hypothesis that seed decay pathogens elicit intricate and dynamic biochemical responses in dormant seeds.

Biological Control of Botrytis cinerea: Interactions with Native Vineyard Yeasts from Washington State.

Native yeasts are of increasing interest to researchers, grape growers, and vintners because of their potential for biocontrol activity and their contributions to the aroma, flavor, and mouthfeel qualities of wines. To assess biocontrol activity, we tested 11 yeasts from Washington vineyards, representing isolates of Candida saitoana, Curvibasidium pallidicorallinum, Metschnikowia chrysoperlae, M. pulcherrima, Meyerozyma guilliermondii, Saccharomyces cerevisiae, and Wickerhamomyces anomalus, for ability to colonize Thompson Seedless grape berries, inhibit the growth of Botrytis cinerea in vitro, and suppress disease symptoms on isolated berries. The yeast-like fungus Aureobasidium pullulans was also included based on its known biocontrol activity against B. cinerea in studies on apple and grape. All yeast strains multiplied rapidly in grape berries and reached densities of over log 6 cells per wound as early as 2 days after inoculation with 200 cells. One of the Botrytis isolates used in this study was much less virulent than the others and was provisionally identified as B. prunorum based on multilocus sequence analysis. Suppression of the growth of B. cinerea isolates 111bb, 207a, 207cb, and 407cb occurred on berries treated with A. pullulans P01A006, Metschnikowia chrysoperlae P34A004 and P40A002, M. pulcherrima P01A016 and P01C004, Meyerozyma guilliermondii P34D003, and S. cerevisiae HNN11516. Inhibition of Botrytis isolates by the yeast strains was more common on berries than in vitro, suggesting the possibility that niche competition was a more likely biocontrol mechanism than antibiosis in planta. Metabolic profiling of yeast strains and B. cinerea isolates using Biolog YT plates revealed seven distinct metabolic groups. Furthermore, the yeast strains showed partial to complete tolerance to the commonly used fungicides fluopyram, triflumizole, metrafenone, pyraclostrobin, and boscalid. Implications of these findings for field deployment of native Washington yeasts as biocontrol agents against B. cinerea are discussed.

Rapid Quantitative Assessment of Rhizoctonia Resistance in Roots of Selected Wheat and Barley Genotypes.

Rhizoctonia solani AG8, causal agent of Rhizoctonia root rot and bare patch in dryland cereal production systems of the Pacific Northwest United States and Australia, reduces yields in a wide range of crops. Disease is not consistently controlled by available management practices, so genetic resistance would be a desirable resource for growers. In this report, we describe three rapid and low-cost assays for R. solani AG8 resistance in wheat and barley, with the view of facilitating screens for genetic resistance in these hosts. The first assay uses 50-ml conical centrifuge tubes containing soil infested with R. solani AG8 on a substrate of ground oats. The second assay uses roots of 3-day-old seedlings directly coated with infested ground oats, followed by incubation in plastic dishes. The third assay, suitable for barley, uses whole infested oat kernels in 50-ml tubes. Symptoms are quantified on the bases of root fresh weight and total root length at 7 and 3 days for the tube and coating assays, respectively. Each of the assays show the same disease differential between susceptible and partially resistant wheat genotypes. The assays can be conducted in the laboratory, growth chamber, or greenhouse.

Agroecological factors correlated to soil DNA concentrations of Rhizoctonia in dryland wheat production zones of Washington state, USA.

The necrotrophic soilborne fungal pathogens Rhizoctonia solani AG8 and R. oryzae are principal causal agents of Rhizoctonia root rot and bare patch of wheat in dryland cropping systems of the Pacific Northwest. A 3-year survey of 33 parcels at 11 growers' sites and 60 trial plots at 12 Washington State University cereal variety test locations was undertaken to understand the distribution of these pathogens. Pathogen DNA concentrations in soils, quantified using real-time polymerase chain reaction, were correlated with precipitation, temperature maxima and minima, and soil texture factors in a pathogen-specific manner. Specifically, R. solani AG8 DNA concentration was negatively correlated with precipitation and not correlated with temperature minima, whereas R. oryzae concentration was correlated with temperature minima but not with precipitation. However, both pathogens were more abundant in soils with higher sand and lower clay content. Principal component analysis also indicated that unique groups of meteorological and soil factors were associated with each pathogen. Furthermore, tillage did not affect R. oryzae but affected R. solani AG8 at P = 0.06. Lower soil concentrations of R. solani AG8 but not R. oryzae occurred when the previously planted crop was a broadleaf (P < 0.05). Our findings showed that R. solani AG8 concentrations were consistent with the general distribution of bare patch symptoms, based on field observations and surveys of other pathogens, but was present at many sites in which bare patch symptoms were not evident. Management of Rhizoctonia root rot and bare patch should account for the likelihood that each pathogen is affected by a unique group of agroecological variables.

Species-Specific PCR Assays for Differentiating Heterodera filipjevi and H. avenae.

Heterodera avenae and H. filipjevi are economically important cyst nematodes that restrict production of cereal crops in the Pacific Northwest United States and elsewhere in the world. Identification of these two species is critical for recommending and implementing effective management practices. Primers were designed from the internal transcribed spacer (ITS) regions of H. avenae and H. filipjevi ribosomal DNA. The primers were highly specific when examined on target isolates but did not amplify DNA from nontarget Heterodera, Globodera, Meloidogyne, Pratylenchus, and other nematode species tested. Polymerase chain reaction (PCR) and amplification conditions were established, and H. avenae and H. filipjevi were clearly distinguished by PCR fragments of 242 and 170 bp, respectively. Robust PCR amplification was achieved with DNA extracted from a single egg or second-stage juvenile (J2) using a laboratory-made worm lysis buffer, and DNA from 0.5 egg or J2 using a commercial kit. The PCR assays were successfully employed for differentiation of H. filipjevi and H. avenae populations collected from eight locations in three Pacific Northwest states. This is the first report of a species-specific ITS PCR assay to detect and identify H. filipjevi. The assays for both species will enhance diagnosis of cereal cyst nematode species in infested fields.

Developing a Real-Time PCR Assay for Detection and Quantification of Pratylenchus neglectus in Soil.

Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R2 = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R2 = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.

Molecular Detection and Quantification of Pythium Species: Evolving Taxonomy, New Tools, and Challenges.

The genus Pythium is one of the most important groups of soilborne plant pathogens, present in almost every agricultural soil and attacking the roots of thousands of hosts, reducing crop yield and quality. Most species are generalists, necrotrophic pathogens that infect young juvenile tissue. In fact, Cook and Veseth have called Pythium the "common cold" of wheat, because of its chronic nature and ubiquitous distribution. Where Pythium spp. are the cause of seedling damping-off or emergence reduction, the causal agent can easily be identified based on symptoms and culturing. In more mature plants, however, infection by Pythium spp. is more difficult to diagnose, because of the nonspecific symptoms that could have abiotic causes such as nutrient deficiencies or be due to other root rotting pathogens. Molecular methods that can accurately identify and quantify this important group are needed for disease diagnosis and management recommendations and to better understand the epidemiology and ecology of this important group. The purpose of this article is to outline the current state-of-the-art in the detection and quantification of this important genus. In addition, we will introduce the reader to new changes in the taxonomy of this group.

Virus-induced gene silencing (VIGS) of genes expressed in root, leaf, and meiotic tissues of wheat.

Barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in wheat leaves, but its utility to transiently express genes, and silencing in other tissues including root, flower, and developing grains, has not been demonstrated in monocots. We monitored green fluorescent protein (GFP) expression to demonstrate the utility of BSMV as a transient expression vector and silenced genes in various wheat tissues to expand VIGS utility to characterize tissue-specific genes. An antisense construct designed for coronatine insensitive1 (COI1) showed an 85% decrease in COI1 transcript level in roots accompanied by a 26% reduction in root length. Similarly, silencing of seed-specific granule-bound starch synthase by antisense and hairpin constructs resulted in up to 82% reduction in amylose content of the developing grains. VIGS of meiosis-specific genes demonstrated by silencing wheat homologue of disrupted meiosis cDNA1 (DMC1) by an antisense construct resulted in a 75-80% reduction in DMC1 transcript level accompanied by an average of 37.2 univalents at metaphase I. The virus-based transient GFP expression was observed in the leaf, phloem, and root cortex at 10-17 days post-inoculation. A novel observation was made that 8-11% of the first selfed generation progeny showed VIGS inheritance and that this proportion increased to 53-72% in the second and to 90-100% in the third generations. No viral symptoms were observed in the progeny, making it possible to study agronomic traits by VIGS. VIGS inheritance is particularly useful to study genes expressing during seed germination or other stages of early plant growth.

Detection and quantification of Pratylenchus thornei in DNA extracted from soil using real-time PCR.

The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.

Native yeast and non-yeast fungal communities of Cabernet Sauvignon berries from two Washington State vineyards, and persistence in spontaneous fermentation.

To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar 'Cabernet Sauvignon' from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.

Real-time PCR quantification of Rhizoctonia solani AG-3 from soil samples.

Rhizoctonia solani anastomosis group 3 (AG-3) causes several diseases of potato, including black scurf and stem canker, affecting potato production in the Skagit Valley, Washington, and around the world. Primers for a SYBR-Green II-based real-time polymerase chain reaction (qPCR) assay were designed from sequences of the nuclear internal transcribed spacer (ITS) regions of fungal isolates of potato and onion from the Pacific Northwest, USA. The primers preferentially amplified R. solani AG-3 DNA, compared to DNA from R. solani AG-4, AG-5 and AG-8. In silico analysis of primer-template duplex stability indicated that the assay also will detect R. solani AG-3 isolates from pea and onion in Washington State and from diverse crop species around the world, but not R. solani AG-9 and AG-2-1. The assay was used to quantify R. solani AG-3 populations in pathogen-infested field soils after temporary flooding rotation, a practice found to be effective for reducing Sclerotinia sclerotiorum and R. solani AG-3 in potatoes in growth chamber studies. Population densities of the pathogen were not significantly reduced in saturated (flooded) soils relative to fallow. However, the qPCR approach was more sensitive and quantitative than the toothpick baiting method for diagnosis of these soil samples. Accurate detection and quantification of R. solani AG-3 in soil will facilitate the development of integrated management plans for Rhizoctonia diseases of potato.

Purinoceptor P2K1/DORN1 Enhances Plant Resistance Against a Soilborne Fungal Pathogen, Rhizoctonia solani.

The purinoceptor P2K1/DORN1 recognizes extracellular ATP, a damage-associated molecular pattern (DAMP) released upon cellular disruption by wounding and necrosis, which in turn, boost plant innate immunity. P2K1 is known to confer plant resistance to foliar biotrophic, hemi-biotrophic, and necrotrophic pathogens. However, until now, no information was available on its function in defense against root pathogens. In this report, we describe the contribution of P2K1 to resistance in Arabidopsis against Rhizoctonia solani, a broad host range, necrotrophic soilborne fungal pathogen. In pot assays, the Arabidopsis P2K1 overexpression line OxP2K1 showed longer root length and a greater rosette surface area than wild type in the presence of the pathogen. In contrast, the knockout mutant dorn1-3 and the double mutant rbohd/f, defective in two subunits of the respiratory burst complex NADPH oxidase, exhibited significant reductions in shoot and root lengths and rosette surface area compared to wild type when the pathogen was present. Expression of PR1, PDF1.2, and JAZ5 in the roots was reduced in dorn1-3 and rbohd/f and elevated in OxP2K1 relative to wild type, indicating that the salicylate and jasmonate defense signaling pathways functioned in resistance. These results indicated that a DAMP-mediated defense system confers basal resistance against an important root necrotrophic fungal pathogen.

Real-time PCR assays for the quantification of native yeast DNA in grape berry and fermentation extracts.

Native yeasts comprise part of the microbial community in grape vineyards and play roles in alcoholic fermentation and wine quality. Monitoring populations of native yeast in vineyards, during fermentation and after bottling will provide viticulturalists and oenologists with information needed to help control spoilage and to enhance desirable wine properties. This is especially crucial for low-intervention winemaking, in which fermentation is driven by native rather than starter microbes. In this study, we report real-time polymerase chain reaction (qPCR) assays for rapid quantification of seven grape yeast species or species combinations that occur in vineyards of Washington State and throughout the world. The assays targeted Candida californica, Curvibasidium pallidicorallinum, Metschnikowia spp., Meyerozyma caribbica/Me. guilliermondii, and Saccharomyces cerevisiae/S. bayanus. We also developed assays for the spoilage yeast Brettanomyces bruxellensis, and the yeast-like fungus Aureobasidium pullulans. Primers were designed for sequences in the internal transcribed spacer (ITS) and large ribosome subunit (LSU) gene. Known populations of yeast cells, added to fermentation extract, were significantly correlated to amounts of purified DNA in picograms (pg) for most of the yeasts; exceptions were A. pullulans and Cu. pallidicorallinum. The utility of the Metschnikowia, Meyerozyma and Saccharomyces assays was further validated by good correlations (R2 = 0.75-0.83) between the number of target sequences and pg of DNA from qPCR for selected vineyard and fermentation samples. Overall, the assays will aid in species identification and monitoring of specific yeasts from cultures, vineyards and fermentation samples. Topics: Food Microbiology, Microbiological Method.

Chronic Sublethal Aluminum Exposure and Avena fatua Caryopsis Colonization Influence Gene Expression of Fusarium avenaceum F.a.1.

Fusarium avenaceum F.a.1 is a novel strain of a fungal plant pathogen capable of preferentially decaying wild oat (Avena fatua) caryopses compared with those of wheat (Triticum aestivum). Understanding the molecular mechanisms governing weed seed-pathogen interactions is crucial to developing novel weed seed suppression technologies. Additionally, wild oat often competes with wheat in regions undergoing soil acidification, which leads to increases in soluble concentrations of many metals, including aluminum (Al). There is a dearth of information regarding the gene expression responses of Fusarium species to Al toxicity, or how metal toxicity might influence caryopsis colonization. To address this, a transcriptomic approach was used to investigate molecular responses of F.a.1 during wild oat caryopsis colonization in the presence and absence of chronic, sublethal concentrations of Al (400 µM). Caryopsis colonization was associated with induction of genes related to virulence, development, iron metabolism, oxidoreduction, stress, and detoxification, along with repression of genes associated with development, transport, cell-wall turnover, and virulence. Caryopsis colonization during Al exposure resulted in the induction of genes associated with virulence, detoxification, stress, iron metabolism, oxidoreduction, and cell wall turnover, along with repression of genes associated with cell wall metabolism, virulence, development, detoxification, stress, and transcriptional regulation. Aluminum exposure in the absence of caryopses was associated with induction of genes involved in siderophore biosynthesis, secretion, uptake, and utilization, along with several other iron metabolism-related and organic acid metabolism-related genes. The siderophore-related responses associated with Al toxicity occurred concurrently with differential regulation of genes indicating disruption of iron homeostasis. These findings suggest Al toxicity is attenuated by siderophore metabolism in F.a.1. In summary, both caryopsis colonization and Al toxicity uniquely influence transcriptomic responses of F.a.1.

Real-time PCR quantification of Fusarium avenaceum in soil and seeds.

The pathogenic fungus Fusarium avenaceum infects a broad range of plant hosts across the globe. While primarily soilborne, F. avenaceum can colonize all plant tissues, including buds, seeds, fruits, stems, crowns, and roots, resulting in significant crop yield reductions and economic losses for growers. In addition to its impact on crop productivity, F. avenaceum produces toxic metabolites that can be transferred to humans and livestock through consumption of infected foods. The ability of F. avenaceum to cause seed decay may be utilized to deplete the weed seedbank in soil, an important integrated weed management strategy. We developed a SYBR Green I-based real-time polymerase chain reaction (qPCR) assay to efficiently detect and quantify F. avenaceum in soil, wild oat (Avena fatua L.) seed caryopses, and wild oat seed hulls. The primer pair was designed from the translation elongation factor 1-alpha (TEF1) gene. In silico and wet lab testing were done to assess the ability of the primers to bind TEF1 sequences from Fusarium spp. and common soil fungi. The findings indicated that the primers were specific to F. avenaceum, and also recognized GenBank TEF1 accessions annotated as F. arthrosporioides, which has been listed as a foliar pathogen of wheat in Oregon, and conspecific with F. avenaceum. Standard curves of F. avenaceum DNA diluted with soil, caryopsis, or hull extracts indicated primer amplification efficiency was not significantly affected by PCR inhibitors. This real-time PCR assay effectively assesses the presence and abundance of F. avenaceum and its close relative F. arthrosporioides, if present, in soil and seed tissues. The assay can be used for endpoint PCR as well.

Detection and Discrimination of Pratylenchus neglectus and P. thornei in DNA Extracts from Soil.

A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei from soil. A primer set was designed from Pratylenchus 28S rRNA gene sequences of the D3 expansion domain. Primer specificity was confirmed with 23 isolates of 15 nematode species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and a method developed in our laboratory gave comparable amplification. PCR conditions were optimized and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy and morphology, and could be used as a rapid diagnostic tool for commercial and research applications for disease forecasting and management.

Corrigendum: An In vitro Study of Bio-Control and Plant Growth Promotion Potential of Salicaceae Endophytes.

[This corrects the article DOI: 10.3389/fmicb.2017.00386.].

Defense Enzyme Responses in Dormant Wild Oat and Wheat Caryopses Challenged with a Seed Decay Pathogen.

Seeds have well-established passive physical and chemical defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. However, there are few studies evaluating potential biochemical defenses of dormant seeds against pathogens. Caryopsis decay by the pathogenic Fusarium avenaceum strain F.a.1 was relatively rapid in wild oat (Avena fatua L.) isoline "M73," with >50% decay after 8 days with almost no decay in wheat (Triticum aestivum L.) var. RL4137. Thus, this fungal strain has potential for selective decay of wild oat relative to wheat. To study defense enzyme activities, wild oat and wheat caryopses were incubated with F.a.1 for 2-3 days. Whole caryopses were incubated in assay reagents to measure extrinsic defense enzyme activities. Polyphenol oxidase, exochitinase, and peroxidase were induced in whole caryopses, but oxalate oxidase was reduced, in response to F.a.1 in both species. To evaluate whether defense enzyme activities were released from the caryopsis surface, caryopses were washed with buffer and enzyme activity was measured in the leachate. Significant activities of polyphenol oxidase, exochitinase, and peroxidase, but not oxalate oxidase, were leached from caryopses. Defense enzyme responses were qualitatively similar in the wild oat and wheat genotypes evaluated. Although the absolute enzyme activities were generally greater in whole caryopses than in leachates, the relative degree of induction of polyphenol oxidase, exochitinase, and peroxidase by F.a.1 was greater in caryopsis leachates, indicating that a disproportionate quantity of the induced activity was released into the environment from the caryopsis surface, consistent with their assumed role in defense. It is unlikely that the specific defense enzymes studied here play a key role in the differential susceptibility to decay by F.a.1 in these two genotypes since defense enzyme activities were greater in the more susceptible wild oat, compared to wheat. Results are consistent with the hypotheses that (1) dormant seeds are capable of mounting complex responses to pathogens, (2) a diversity of defense enzymes are involved in responses in multiple plant species, and (3) it is possible to identify fungi capable of selective decay of weed seeds without damaging crop seeds, a concept that may be applicable to weed management in the field. While earlier work on seed defenses demonstrated the presence of passive defenses, this work shows that dormant seeds are also quite responsive and capable of activating and releasing defense enzymes in response to a pathogen.

An In vitro Study of Bio-Control and Plant Growth Promotion Potential of Salicaceae Endophytes.

Microbial communities in the endosphere of Salicaceae plants, poplar (Populus trichocarpa) and willow (Salix sitchensis), have been demonstrated to be important for plant growth promotion, protection from biotic and abiotic stresses, and degradation of toxic compounds. Our study aimed to investigate bio-control activities of Salicaceae endophytes against various soil borne plant pathogens including Rhizoctonia solani AG-8, Fusarium culmorum, Gaeumannomyces graminis var. tritici, and Pythium ultimum. Additionally, different plant growth promoting traits such as biological nitrogen fixation (BNF), indole-3-acetic acid (IAA) biosynthesis, phosphate solubilization, and siderophore production were assessed in all bio-control positive strains. Burkholderia, Rahnella, Pseudomonas, and Curtobacterium were major endophyte genera that showed bio-control activities in the in-vitro assays. The bio-control activities of Burkholderia strains were stronger across all tested plant pathogens as compared to other stains. Genomes of sequenced Burkholderia strains WP40 and WP42 were surveyed to identify the putative genes involved in the bio-control activities. The ocf and hcnABC gene clusters responsible for biosynthesis of the anti-fungal metabolites, occidiofungin and hydrogen cyanide, are present in the genomes of WP40 and WP42. Nearly all endophyte strains showing the bio-control activities produced IAA, solubilized tricalcium phosphate, and synthesized siderophores in the culture medium. Moreover, some strains reduced acetylene into ethylene in the acetylene reduction assay, a common assay used for BNF. Salicaceae endophytes could be useful for bio-control of various plant pathogens, and plant growth promotion possibly through the mechanisms of BNF, IAA production, and nutrient acquisition.