Fungal endophytes enhance wheat heat and drought tolerance in terms of grain yield and second-generation seed viability.

AIMS: We evaluated the impact of fungal endophyte symbiosis on the growth, ecophysiological and reproductive success of wheat exposed to heat and drought. METHODS AND RESULTS: The resistance of pot-grown wheat to heat or drought stress was measured by quantifying efficiency of photosystem II (Fv /Fm), plant height, average seed weight (ASW), total seed weight (TSW), water-use efficiency (WUE) as well as time to 50% germination and percentage germination of second-generation seeds produced under heat stress, drought stress or well-watered conditions. The endophytic fungi tested increased wheat tolerance for drought and heat. Endophyte SMCD 2206 was the most beneficial, followed by SMCD 2210 and 2215. Surprisingly, second-generation seeds produced by drought-stressed wheat colonized by SMCD 2206, 2210 or 2215 had decreased WUE relative to those produced by endophyte-free, drought-stressed plants. However, these seeds germinated more rapidly than those produced by endophyte-free, stressed parental plants. CONCLUSIONS: The tested consortium of endophytes has the potential to improve wheat adaptation to heat and drought. SIGNIFICANCE AND IMPACT OF THE STUDY: The capacity of endophytes to increase wheat tolerance for abiotic stress and to improved germination in endophyte-free second-generation seeds arising from stressed plants could be applicable to agriculture. The mechanisms by which intergenerational endophyte-mediated affects occurs warrant further research.

Nontarget effects of foliar fungicide application on the rhizosphere: diversity of nifH gene and nodulation in chickpea field.

AIMS: This study explores nontarget effects of fungicide application on field-grown chickpea. METHODS AND RESULTS: Molecular methods were used to test the effects of foliar application of fungicide on the diversity and distribution of nifH genes associated with two chickpea cultivars and their nodulation. Treatments were replicated four times in a split-plot design in the field, in 2008 and 2009. Chemical disease control did not change the richness of the nifH genes associated with chickpea, but selected different dominant nifH gene sequences in 2008, as revealed by correspondence analysis. Disease control strategies had no significant effect on disease severity or nifH gene distribution in 2009. Dry weather conditions rather than disease restricted plant growth that year, suggesting that reduced infection rather than the fungicide is the factor modifying the distribution of nifH gene in chickpea rhizosphere. Reduced nodule size and enhanced N(2) -fixation in protected plants indicate that disease control affects plant physiology, which may in turn influence rhizosphere bacteria. The genotypes of chickpea also affected the diversity of the nifH gene in the rhizosphere, illustrating the importance of plant selective effects on bacterial communities. CONCLUSIONS: We conclude that the chemical disease control affects nodulation and the diversity of nifH gene in chickpea rhizosphere, by modifying host plant physiology. A direct effect of fungicide on the bacteria cannot be ruled out, however, as residual amounts of fungicide were found to accumulate in the rhizosphere soil of protected plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Systemic nontarget effect of phytoprotection on nifH gene diversity in chickpea rhizosphere is reported for the first time. This result suggests the possibility of manipulating associative biological nitrogen fixation in the field.

Assessment of alcohol percentage test for fungal surface hydrophobicity measurement.

AIM: To determine whether assessing the penetration of solutions with different concentrations of ethanol (alcohol percentage test: APT) on fungal surfaces is effective in characterization of hydrophobicity on fungal surfaces. METHODS AND RESULTS: APT and contact angle (CA) measurements were conducted on nine hydrophobic and two hydrophilic fungal strains from the phyla of Ascomycota, Basidiomycota and Zygomycota. There was a strong positive correlation (R(2) = 0.95) between the APT and CA measurements from eight of the nine hydrophobic stains (four pathogenic and mycotoxigenic Fusarium taxa, one melanosporaceous biotrophic taxon, Alternaria sp, Penicillium aurantiogriseum and Cladosporium cladosporioides). Hydrophilic control strains, Mortierella hyalina and Laccaria laccata, had CAs <90 degrees and no measurable degree of hydrophobicity using the APT method. CONCLUSIONS: The APT method was effective in measuring the degree of hydrophobicity and can be conducted on different zones of fungal growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of fungal surface hydrophobicity is important for understanding of its particular role and function in fungal morphogenesis and pathogenesis. APT is a simple method that can be utilized for fungal hydrophobicity measurements when CA cannot be measured because of obscured view from aerial mycelia growth.

Whole-cell protein and ITS rDNA profiles as diagnostic tools to discriminate Fusarium avenaceum intraspecific variability and associated virulence.

A total of 91 isolates of Fusarium avenaceum were regrouped into 15 phenotypes and 10 vegetative compatibility groups showing specific one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1-D SDS-PAGE) protein profiles and less-specific internal transcribed spacer rDNA profiles. Each isolate possessed reproducible signature protein bands. Indeed, the unweighted pair group method with arithmetic averages clustering revealed that the protein profile of each group of isolates correlated with fungus virulence. The use of SDS-PAGE offers a simple and sensitive technique for routine differentiation between pathogenic and nonpathogenic isolates within unknown F. avenaceum populations. The discovery has significant implications for risk assessment of cereal yield to ensure food and feed safety. This low-cost approach has the potential to be optimized and extended to a broad spectrum of Fusarium head blight pathogens.

Fungal diversity, dominance, and community structure in the rhizosphere of clonal Picea mariana plants throughout nursery production chronosequences.

Fungal diversity in the rhizosphere of healthy and diseased clonal black spruce (Picea mariana) plants was analyzed with regard to nursery production chronosequences. The four key production stages were sampled: mother plants (MP), 8-week-old cuttings (B + 0), second-year cuttings (B + 1), and third-year cuttings (B + 2). A total of 45 fungal taxa were isolated and identified based on cultural, phenotypic, and molecular characters. Members of phylum Ascomycota dominated, followed by Basidiomycota and Zygomycota. Diagnosis characters and distance analysis of the internal transcribed spacer rDNA sequences allowed the identification of 39 ascomycetous taxa. Many belong to the order Hypocreales, families Hypocreaceae and Nectriaceae, which contain many clusters of potentially pathogenic taxa (Cylindrocladium, Fusarium, and Neonectria) and are also ecologically associated with antagonistic taxa (Chaetomium, Hypocrea, Microsphaeropsis, Penicillium, Paecilomyces, Verticillium, Trichoderma, and Sporothrix). This is also the first report of a Cylindrocladium canadense association with disease symptoms and relation with Pestalotiopsis, Fusarium, Exserochilum, Rhizoctonia, and Xenochalara fungal consortia. Both production chronosequence and plant health considerably influenced fungal taxa assemblages. Unweighted pair-group arithmetic average clustering showed that isolates from MP, B + 0, and B + 1 plant rhizospheres clustered together within healthy or diseased health classes, whereas isolates from healthy and diseased B + 2 plants clustered together. Canonical correspondence analysis revealed substantial alteration in community assemblages with regard to plant health and yielded a principal axis direction that regrouped taxa associated with diseased plant rhizosphere soil, whereas the opposite axis direction was associated with healthy plants. Two diversity indices were defined and applied to assess the fungal taxa contribution (Tc) and persistence (Pi) throughout the production.

First Report of Damping-Off of Durum Wheat Caused by Arthrinium sacchari in the Semi-Arid Saskatchewan Fields.

Durum wheat (Triticum durum Desf.) is an important crop in western Canada. In 2005, Arthrinium sacchari was frequently isolated from soil and durum wheat plants of the semi-arid fields of Swift Current, Saskatchewan, Canada (50°16'N, 107°44'W). The susceptibility of durum wheat to damping-off caused by this fungus was evaluated. To our knowledge, this is the first report of A. sacchari in North America (1) and the first mention of its association with durum wheat. DNA was extracted (MoBio Isolation Kit, Carlsbad, CA) from 2-week-old A. sacchari isolates (FBC.3, FBC.45, and FBC.143) grown on PDA. The internal transcribed spacer (ITS) of the rDNA was amplified from each isolate and sequenced (Plant Biotechnology Institute, Saskatoon, SK, Canada) and similarity analyses were performed using the BLAST search algorithm in GenBank. All three sequences (Accession Nos. EF076710, EF076711, and EF076712A) showed 99% similarity with A. sacchari (Accession No. AF393679). An in vitro assay was performed by placing 1-cm2 agar plugs containing mycelia of A. sacchari (FBC.3, FBC.45, and FBC.143) onto surface-sterilized durum. Surface-sterilized seeds inoculated in the same way with Fusarium graminearum or F. avenaceum were used as negative controls, and noninoculated surface-sterilized seeds were used as a positive control. A second in vitro assay involved inoculating the same isolates onto seeds placed in sterilized sandy soil. In both assays, 10 seeds per petri plate and three plates per treatment were used and plates were incubated at 21°C for 1 week in darkness. All experiments were performed twice. On PDA, preemergence damping-off was found in 60% of A. sacchari FBC.3, 55% of A. sacchari FBC.45 and FBC.143, 50% of F. avenaceum, and 58% of F. graminearum inoculated seeds. In sterilized soil, the incidence of preemergence damping-off ranged from 43 to 30%. Subsequent incubation over a period of 3 weeks resulted in 100% postemergence damping-off in A. sacchari FBC.45 and FBC.3 as well as in both Fusarium spp. inoculated controls, 60% postemergence damping-off in A. sacchari FBC.143, and no damping-off in the noninoculated control. Arthrinium and Fusarium spp. were reisolated only from symptomatic plants, satisfying Koch's postulates. In conclusion, durum wheat is highly susceptible to damping-off caused by A. sacchari, showing characteristic dark brown or violet lesions in infected tissues. A. sacchari was previously reported to be present in South America and eastern Asia. In China, it is considered an important mycotoxigenic species (2). Thus, infection of durum wheat crops with A. sacchari could pose a significant threat to North American wheat production. References: (1) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory. Online publication. ARS, USDA, 2006. (2) X. J. Liu et al. Acta Mycol. Sinica 7:221, 1988.

Root Rot of Black Spruce Caused by Cylindrocladium canadense in Eastern North America.

During October 2002, symptoms of root rot of black spruce, Picea mariana (Mill.) B.S.P., were observed in the St-Modeste (47°46'N, 69°36'W) conifer nursery (400 km northeast of Montreal, Quebec, Canada). Disease severity was low in the greenhouse-produced mother plants and 1-year-old seedlings and moderate in field-grown 2- and 3-year-old seedlings. A species of Cylindrocladium was isolated on potato dextrose agar from 12 symptomatic seedlings from the greenhouse and 12 from the field. The isolates produced chestnut-colored colonies and chlamydospores, both of which were typical of C. canadense Kang, Crous & Schoch (2). DNA was extracted from representative isolates (MTF 101, MTF 102), and the internal transcribed spacer (ITS) of the rDNA gene was amplified and sequenced (GenBank Accession Nos. AY705980 and AY705981). There was a 99% match with a sequence of C. canadense (GenBank Accession No. AF348256). However, there was approximately 10% divergence with the ITS sequence of C. floridanum (GenBank Accession No AF307343). MTF101 and MTF102 were pathogenic on black spruce seedlings when fungal suspension (106 CFU/ml) was added to germinating seeds in petri plates or infiltrated into roots of 2-week-old seedlings growing in sterilized, moist, sandy soil in the greenhouse. Within 3 weeks, inoculated seedlings exhibited typical root necrosis, while control seedlings were symptomless. C. canadense was reisolated only from symptomatic seedlings. The occurrence of C. canadense in eastern North America has significant implications for forestry regeneration. Previously, only C. floridanum had been reported as pathogenic in the St-Modeste nursery and in eastern North America(1). References: (1) R. C. Hamelin et al. Appl. Environ. Microbiol. 62:4026, 1996. (2) J. C. Kang et al. Syst. Appl. Microbiol. 24:206, 2001.

A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus.

In North America, asparagus (Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani, clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples.

First Report of Root Rot on Asparagus Caused by Phytophthora megasperma in Canada.

In August 2002, Phytophthora megasperma Drechs. was isolated from wilted plants of Asparagus officinalis L. cv. Guelph Millenium displaying spear and crown rot. Six affected plants were sampled in a commercial asparagus field located in the Saguenay-Lac-Saint-Jean Region (300 km northeast of Montreal, Quebec, Canada). The fungus was isolated from asparagus fern stalks, crown tissue, and spears after a rainy period and identified using morphological and cultural characteristics (2). In pinkish 4-week-old cultures, unbranched stalks bore abundant sporangia, which were ovoid to obpyriform in shape, 15 to 45 m long, and 10 to 30 m in diameter. Characteristic circular oospores >30 m in diameter were produced on V8 juice agar at 25°C in darkness after 1 month. Pathogenicity was tested on asparagus cvs. Guelph Millenium and Jersey Knight. A mycelium suspension (3 ml at 106 CFU/ml) prepared from 1-week-old shaken potato dextrose (PD) broth was sprayed on 30 1-week-old seedlings grown in petri plates filled with sterilized, moist, sandy soil and held at 20°C (day/night). Controls received sterile PD broth. Within 3 weeks of incubation in the dark, inoculated seedlings exhibited necrotic symptoms similar to those observed initially, while controls remained healthy. The pathogen was isolated from 75% of the 'Guelph Millenium' and 98% of the 'Jersey Knight' symptomatic seedlings, but not isolated from the control seedlings. In North America, disease caused by P. megasperma resulting in yield loss has been reported in California and New York (1,3). In Canada, the etiology of asparagus diseases is not well characterized. To our knowledge, this is the first report of P. megasperma on asparagus plants in Canada. References: (1) P. A. Ark and J. T. Barrett. Phytopathology 28:754, 1938. (2) D. C. Erwin et al. Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology. The American Phytopathological Society, St Paul, MN, 1983. (3) T-L. Kuan and D. C. Erwin. Phytopathology 70:333, 1980.

Airborne fungal ecological niche determination as one of the possibilities for indirect mycotoxin risk assessment in indoor air.

Based on the microbiological analysis of air samples from occupied spaces, some possibilities for indirect risk assessment of mycotoxin-related health problems are proposed. Airborne fungi could be classified on the basis of the relationship between the two environmental factors and their combinations, i.e., temperature and water requirements (water activity aw). One type involves three different groups of molds, selected on the basis of the quantitative and qualitative information about the ability of fungi to sporulate under different environmental conditions: group (i), represented by Aspergillus nidulans, A. niger, and A. ochraceus, and characterized by sporulation which was more dependent on temperature than on water activity; (ii), represented by A. flavus and A. versicolor, in which sporulation was approximately equal and depended on both the temperature changes and aw alterations; and (iii), represented by Cladosporium sp., Penicillium cyclopium, and P. citrinum, in which sporulation depended more on alteration of the aw conditions than on temperature changes. Another type is characterized by four sporulation rates with two levels of mycotoxin risk accumulation in the spores (conidia) of each mold species: large (Ia) and moderate (Ib) sporulation rates with a risk of mycotoxin accumulation (aw > or = 86; t > or = 12 degrees C); rare sporulation (IIa) and absence of sporulation (IIb), without risk of mycotoxin accumulation (aw < or = 86; t < or = 12 degrees C). In conclusion, providing a useful guide for two dimensions, temperature and water activity for each of the three phases of fungal growth, i.e. germination, growth, and sporulation, could be important for determination of the fundamental niche of each fungus and its ability to form or accumulate mycotoxin. Special emphasis should be given to the indirect mycotoxin risk assessment in heating, ventilation, and air conditioning systems.