Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis

Fusarium head blight (scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat (Triticum aestivum L.) worldwide. Wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, is one of the major diseases of wheat in China. The defensin RsAFP2, a small cyteine-rich antifungal protein from radish (Raphanus sativus), was shown to inhibit growth in vitro of agronomically important fungal pathogens, such as F. graminearum and R. cerealis. The RsAFP2 gene was transformed into Chinese wheat variety Yangmai 12 via biolistic bombardment to assess the effectiveness of the defensin in protecting wheat from the fungal pathogens in multiple locations and years. The genomic PCR and Southern blot analyses indicated that RsAFP2 was integrated into the genomes of the transgenic wheat lines and heritable. RT-PCR and Western blot proved that the RsAFP2 was expressed in these transgenic wheat lines. Disease tests showed that four RsAFP2 transgenic lines (RA1–RA4) displayed enhanced resistance to F. graminearum compared to the untransformed Yangmai 12 and the null-segregated plants. Assays on Q-RT-PCR and disease severity showed that the express level of RsAFP2 was associated with the enhanced resistance degree. Two of these transgenic lines (RA1 and RA2) also exhibited enhanced resistance to R. cerealis. These results indicated that the expression of RsAFP2 conferred increased resistance to F. graminearum and R. cerealis in transgenic wheat.     

Variability and characterization of mycotoxin-producing Fusarium spp isolates by PCR-RFLP analysis of the IGS-rDNA region

In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB₁, FB₂, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rDNA. The results indicated that G. fujikuroi complex isolates can be␣divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA␣and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.     

The effect of toxin-producing Fusarium fungi on behavior of the rice weevil Sitophilus oryzae (Coleoptera,Dryophthoridae)

The paper describes laboratory tests in which the behavior response of adult rice weevils Sitophilus oryzae L. to the presence of seven species of micromycetes of the genus Fusarium (F. graminearum, F. culmorum, F. cerealis, F. poae, F. sporotrichioides, F. langsethiae, and F. sibiricum; 3 strains for each species) infecting cereals was characterized. The chemicals of unkown structure, released by the fungi, can have both attractive and repellent effects on the weevils; in some cases a neutral response was observed. The strains of Fusarium species characterized as weak pathogens (F. langsethiae, F. poae, and F. sibiricum) in most cases stimulated attractive and neutral responses. Relatively strong pathogens (F. cerealis, F. culmorum, F. graminearum, and F. sporotrichioides) generally had a repellent effect. The results obtained are discussed in the context of possible relationships between Fusarium fungi and the rice weevil during their utilization of cereals as a shared food substrate.     

Diversity of Fusarium Species Isolated from Weeds and Plant Debris in Croatia

Weeds are alternative hosts of plant pathogens and when colonized may not exhibit disease symptoms. In 2008 and 2009, samples of weeds and plant debris were collected from 12 locations in eastern Croatia, and 300 Fusarium isolates colonizing them were identified. Strains were grouped and identified based on morphology and amplified fragment length polymorphism (AFLP) patterns. Portions of the β‐tubulin and translocation elongation factor 1‐α genes were sequenced from representative strains of each group to confirm the identifications. Fourteen Fusarium species were identified with F. graminearum (20%), F. verticillioides (18%), F. oxysporum (16%), F. subglutinans (13%) and F. proliferatum (11%) all present as more than 10% of the population. Fusarium acuminatum, F. avenaceum, F. concolor, F. crookwellense (F. cerealis), F. equiseti, F. semitectum, F. solani, F. sporotrichioides and F. venenatum, were all present at frequencies < 8%. Our results indicate that economically important Fusarium spp. may be isolated from numerous alternative hosts during the off season and that weeds and plant debris can serve as a reservoir of genetically diverse inoculum.     

Potential Biofumigation Effects of Brassica oleracea var. caulorapa on Growth of Fungi

Biofumigation, as an environment‐friendly alternative to methyl bromide is gaining attention in sustainable agricultural production systems. Based on the biofumigation suppression of growth of three soil‐borne filamentous fungi (Fusarium sp., F. oxysporum and P. aphanidermatum), Brassica oleracea var. caulorapa was selected from eight Brassica and other plant species as a potential material for the purpose. Powdered tissues of plants were confined to individual Petri dishes without physical contact with each of the following 28 fungal isolates from 16 hosts: 13 Fusarium spp., two Verticillium dahliae, two Ceratocystis fimbriata, Bipolaris sorokiniana, Gaeumannomyces graminis, Ceratobasidium cornigerum, Rhizotonia cerealis, Phytophthora parasitica, Phytophthora capsici, Botrytis cinerea, two Rhizoctonia solani, Sclerotinia sclerotiorum and Magnaporthe grisea. The level of suppression of growth 7 days after inoculation varied. Based on growth suppression, the 28 isolates were grouped into three clusters by Fuzzy clustering: Cluster I contains F. proliferatum with 20.5% suppression, Cluster II composed of 15 isolates, Fusarium sp., two V. dahliae, two C. fimbriata, B. sorokiniana, C. cornigerum, two R. solani, R. cerealis, S. sclerotiorum, P. parasitica and M. grisea, with 75.2–100% suppression and Cluster III consisted of 12 isolates; five F. oxysporum, F. solani, F. azysporum, F. moniliforme, F. graminearum, F. verticillioides, G. graminis, P. capsici with 40.7–66.2% suppression. Ceratobasidium fimbriata and V. dahliae were more sensitive to biofumigation than S. sclerotiorum and F. culmorum when different amounts of ground powder were used. One gram of powder could suppress the growth of the former two up to 68.6% and 68.7%, but the growth suppression in the latter two by 12.7% and 24.0%, respectively. These results indicated that the amount of plant tissue to be used should be considered depending on target pathogen species. The swollen root of B. oleracea var. botrytis appeared a better material than the leaf for achieving suppression of growth in pathogenic fungi.     

Diversity of the <Emphasis Type="Italic">Fusarium</Emphasis> species associated with head and seedling blight on wheat in Slovakia

Species associated with Fusarium head blight are depending on the production and edaphic conditions. The differences are found in the representation of various Fusarium spp. in the diseases, which sporadically occur all over the territory of Slovakia, in all agricultural production types. We identified fifteen Fusarium species during ten years of investigation. Most of the mentioned species F. culmorum (W.G. Smith) Sacc., F. graminearum Schwabe, recently F. cerealis (Cooke) Sacc. (crookwellense Burgess, Nelson & Tousson) and F. sambucinum Fuckel in diseased caryopsis are seed transmitted. The significant differences among species and intra species in cultural and pathogenicity assays in vitro and in vivo were correlated. Some of them are able to produce toxic metabolites — deoxynivalenone, which probably play a role in the aggressiveness of the pathogen and promote disease development and pathogen colonization.     

Enhancing plant disease suppression by Burkholderia vietnamiensis through chromosomal integration of Bacillus subtilis chitinase gene chi113

Burkholderia vietnamiensis P418 is a plant growth-promoting rhizobacteria. A chitinase gene from Bacillus subtilis was cloned and stably integrated into the chromosome of using the transposon delivery vector, pUTkm1. Chitinase activity was detected in recombinant P418-37 but not in wild type P418. Recombinant P418-37 retained the in vitro growth rate, N₂-fixation and phosphate and potassium-solubilizing characteristics of the wild type. P418-37 significantly (P < 0.05) increased in vitro inhibition of the plant pathogenic fungi Rhizoctonia solani, Fusarium oxysporum f.sp. vasinfectum, Rhizoctonia cerealis, Bipolaris sorokiniana, Verticillium dahliae and Gaeumannomyces graminis var. tritici compared with P418. In planta disease suppression assays indicated that P418-37 significantly (P < 0.05) enhanced suppression of wheat sheath blight (R. cerealis), cotton Fusarium wilt (F. oxysporium f.sp. vasinfectum) and tomato gray mould (Botrytis cinerea), relative to the wild type.     

Wheat-infectingFusarium species in Poland — their chemotypes and frequencies revealed by PCR assay

ThreeFusarium species:F. graminearum, F. culmorum andF. cerealis were identified in laboratory cultures and in sporodochia from spikelets of scabby wheat. SCAR (sequence characterized amplified region) primers were used to identifyFusarium species and nivalenol (NIV) and deoxynivalenol (DON) chemotypes within species in laboratory cultures and field collected heads harvested in 2006. Results from PCR analyses confirmed preliminary identifications of species on the basis of examination of macroconidia under a light microscope and identification of cultures on agar media. NIV and DON (3Ac-DON and 15Ac-DON) chemotypes were identified using PCR assay. Among samples and isolates ofF. graminearum, the 15Ac-DON chemotype dominated, and among those whereF. culmorum was identified, the 3Ac-DON chemotype prevailed. Only 5 of the 41 isolates ofF. graminearum tested, displayed the NIV chemotype. An increase in the frequency ofF. graminearum and a decrease in the frequency ofF. culmorum were found during 1998 to 2006.     

Characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40 with the ability to control wheat sharp eyespot disease

This study details the isolation and characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40, and is the first to examine P. chlororaphis for use in suppression of wheat sharp eyespot on wheat. Pa40 was isolated during an investigation aimed to identify biocontrol agents for Rhizoctonia cerealis. Over 500 bacterial strains were isolated from the rhizosphere of infected wheat and screened for in vitro antibiosis towards R. cerealis and ability to provide biocontrol in planta. Twenty‐six isolates showed highly antagonistic activity towards R. cerealis, in which Pseudomonas spp. and Bacillus spp. were predominant members of the antagonistic community. Strain Pa40 exhibited clear and consistent suppression of wheat sharp eyespot disease in a greenhouse study and suppression was comparable to that of chemical treatment with validamycin A. Pa40 was identified as P. chlororaphis subsp. aurantiaca by the Biolog identification system combined with 16S rDNA, atpD, carA and recA sequence analysis and biochemical and physiological characteristics. To determine broad‐spectrum applicability and the specific mechanisms involved in Pa40's pathogen suppression this strain was tested for antibiosis towards various phytopathogens and assayed for many biocontrol activities and plant‐beneficial traits. Strain Pa40 inhibited the growth of 10 of 13 phytopathogenic fungal strains and six of eight phytopathogenic bacteria tested. This original work characterises HCN, protease and siderophore production in P. chlororaphis. Each of these characteristics likely contributed to Pa40's biocontrol capabilities as well as stimulation of the hypersensitive response in tobacco and the presence of genes involved in the biosynthesis of phenazine, 2‐hydroxylated phenazine and pyrrolnitrin.     

Evaluation of Bacillus amyloliquefaciens PEBA20 as a biocontrol agent for Rhizoctonia cerealis and Bipolaris maydis

Rhizoctonia cerealis and Bipolaris maydis are fungal plant pathogens that cause enormous agricultural losses of wheat and maize.Bacillus amyloliquefaciens PEBA20 inhibited mycelial growth and spore germination of the pathogens and reduced fungal infections in wheat and maize, indicating its potential for application as a biocontrol agent.     

<Emphasis Type="Italic">Cryptococcus randhawai</Emphasis> sp. nov<Emphasis Type="Italic">.,</Emphasis> a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of <Emphasis Type="Italic">Ficus religiosa</Emphasis> (peepal tree) from New Delhi,India

A novel anamorphic Cryptococcus species is described, which was isolated in New Delhi (India) from decaying wood of a tree trunk hollow of Ficus religiosa. On the basis of sequence analysis of the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS)-1 and ITS-2 region sequences, the isolate belonged to the Cryptococcus albidus cluster (Filobasidiales, Tremellomycetes) and was closely related to Cryptococcus saitoi, Cryptococcus cerealis and Cryptococcus friedmannii with 98% sequence identity. Phenotypically, the species differed from C. saitoi with respect to growth temperature (up to 37^oC), presence of a thin capsule, ability to grow in the absence of vitamins, and inability to assimilate citrate and ethylamine. With respect to C. friedmannii, it differed in growth temperature, ability to assimilate lactose, raffinose, l-rhamnose, myo-inositol, and inability to utilize citrate. Furthermore, our isolate also differed from C. cerealis in growth temperature, presence of capsule and inability to assimilate l-sorbose. In view of the above phenotypic differences and unique rDNA sequences, we consider that our isolate represents a new species of Cryptococcus, and therefore, a new species, Cryptococcus randhawai is proposed for this taxon. The type strain J11/2002 has been deposited in the culture collection of the Centraalbureau voor Schimmelcultures (CBS10160) and CABI Biosciences (IMI 393306).     

Cryptococcus cerealis sp. nov. a psychrophilic yeast species isolated from fermented cereals

Two yeast strains isolated in 2007 from fermented pig feed were studied, including the analysis of sequences of the D1/D2 and ITS-regions of the rDNA-repeats, their morphology and nutritional physiology. Sequence comparison of the D1/D2 and ITS regions demonstrated that the strains do not belong to any known species. Therefore, a new species, Cryptococcus cerealis with the type strain CBS 10505, is proposed. The species belongs to Filobasidiales (Agaricomycetes, Basidiomycota), and has Cryptococcus saitoi as the closest related species. The new species is psychrophilic, showing significant growth at 4 and 10°C.     

Detoxification of Corn Antimicrobial Compounds as the Basis for Isolating Fusarium verticillioides and Some Other Fusarium Species from Corn

The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively. Fusarium verticillioides (= F. moniliforme) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29 Fusarium species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were F. verticillioides, F. subglutinans, F. cerealis (= F. crookwellense), and F. graminearum. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of F. verticillioides from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of F. verticillioides infection in field samples.     

Fusarium cerealis Associated with Barley Seeds in Argentina

Fusarium head blight is a fungal disease caused by a complex of Fusarium species on cereals, such as barley and wheat. It has economic impacts due to yield reductions and mycotoxin contamination. As barley production has increased considerably in the last 5 years in Argentina, a survey was conducted for identifying Fusarium species associated with barley grains. Fusarium cerealis was isolated and identified based on morphological and molecular analysis. The potential production of nivalenol and zearalenone was assessed using specific PCR assays. Koch′s postulates were carried out to confirm the pathogenicity of the fungus.     

A STUDY OF THE VITAMIN REQUIREMENTS OF THREE SPECIES OF THE GENUS GELASINOSPORA

Hackbarth , R. D., and R. P. Collins . (U. Connecticut, Storrs.) A study of the vitamin requirements of three species of the genus Gelasinospora. Amer. Jour. Bot. 48(7): 603–606. Illus. 1961.—The vitamin requirements of 3 species of Gelasinospora (G. tetrasperma, G. retispora, G. cerealis) were determined. Various combinations of the 10 vitamins used in the study were tried and biotin was found to be the factor limiting growth.     

Toxigenic Fusarium species infecting wheat heads in Poland

Toxigenic Fusarium species are common pathogens of wheat and other cereals worldwide. In total, 449 wheat heads from six localities in Poland, heavily infected with Fusarium during 2009 season, were examined for Fusarium species identification. F. culmorum was the most common species (72.1% on average) with F. graminearum and F. avenaceum the next most commonly observed, but much less frequent (13.4 and 12.5% respectively). F. cerealis was found in 1.8% of all samples, and F. tricinctum was found only in one sample (0.2%). Subsequent quantification of the three major mycotoxins (deoxynivalenol, zearalenone and moniliformin) in grain and chaff fractions with respect to associated prevailing pathogen species uncovered the following patterns. Moniliformin (MON) was found in low amounts in all samples with F. avenaceum present. In contrast, deoxynivalenol (DON) and zearalenone (ZEA) were the contaminants of F. culmorum- and F. graminearum-infected heads. The highest concentration of DON was recorded in grain sample collected in Radzików (77 µg g^?1). High temperatures in Central Poland during July and August accompanied with high rainfall in July were responsible for this high DON accumulation. Trichothecene, zearalenone, enniatin and beauvericin chemotypes were identified among 21 purified isolates using gene-specific PCR markers.     

Antagonism of Rhizoctonia spp. to Pythium oligandrum and Damping-Off Fungi

In paired cultures on corn-meal agar, Rhizoctonia solani, R. cerealis and R. fragariae caused vacuolation, disappearance of cytoplasm, and apparent lysis of hyphae of Pythium oligandrum, P. ultimum, and Aphanomyces cochlioides. Hyphae of Phoma betae were not injured by the Rhizoctonia spp. When sugar-beet seeds dressed with mycelium of R. cerealis, R. fragariae, or an isolate of R. solani nonpathogenic to sugar-beet were planted in soil naturally infested with P. ul-timum, the level of biological control of damping-off was similar to that obtained with captan dressing. In soil artificially infested with P. ultimum, biological dressings were slightly less efficacious than the chemical dressing.