Isolation and evaluation of a Bacillus methylotrophicus strain for control of corn stalk rot

Bacteria were isolated from corn plants and efficacy of a selected isolate for control of corn stalk rot caused by Fusarium graminearum was evaluated. The bacterial isolate provided the greatest suppression of F. graminearum in lab studies and reduced corn stalk rot significantly in 2013 and 2014 field experiments.     

Identification of a biocontrol agent Bacillus vallismortis BV23 and assessment of effects of its metabolites on Fusarium graminearum causing corn stalk rot

Corn stalk rot, caused by Fusarium graminearum, is one of the most destructive diseases of maize in many regions of the world. A bacterial strain BV23 was isolated from corn rhizosphere that reduced corn stalk rot significantly in greenhouse studies in 2016 and 2017. BV23 was identified as Bacillus vallismortis, which showed antagonistic effects against a number of fungal pathogens, including F. graminearum, Rhizoctonia solani, Athelia rolfsii, and Thanatephorus cucumeris. BV23 had the greatest fungistatic effect on F. graminearum, inhibiting mycelial growth by 66.2%, conidial germination by 90.1%, and conidial production by 86.7%. The probable antifungal mechanism was assessed by examining the morphology and ultrastructure of F. graminearum hyphae. Treatment by BV23 culture supernatant resulted in coarser hyphae, induced cytoplasmic granulation, and increased cell membrane permeability of F. graminearum, causing cytoplasm leakage. These effects became increasingly obvious with increasing concentration (1%, 5% and 10%). Furthermore, the antifungal active substances were sensitive to heat.     

Incidence of Stenocarpella and Fusarium Cob Rots in Monoculture Maize under Different Tillage Systems

The incidence of maize cob rot caused by Stenocarpella maydis, S. macrospora, Fusarium moniliforme, F. subglutinans and F. graminearum was determined over two seasons under different tillage systems at various localities. Tillage had no effect on Fusarium spp. cob rots. S. macrospora occurred only at one locality, viz. Cedara, and no tillage effect was observed. Ploughing reduced the incidence of S. maydis cob rot at localities which had high incidences of disease. The relationship between severity of S. maydis cob rot and surface stubble mass was linear.     

Effect of soil solarization on corn stalk rot

Seven weeks solarization of irrigated soil raised its temperature by 11.5°C over non-solarized soil at 10 cm depth and effectively controlled weeds (98.5%), stalk borer (8.9%) and stalk rot disease (69.1%) in corn. Solarization also reduced symptoms of Fusarium moniliforme and Macrophomina phaseolina significantly by 64.2% and 78.4%, respectively, and completely controlled M. phaseolina in corn cultivars, viz. Pool-10, Shaheen and Gauher. Whereas symptoms of F. moniliforme were observed in these cultivars, Fusarium graminearum was not observed except in two cultivars, Shaheen and Akbar. Growth of crop planted in solarized plots was better and it yielded almost one to three times more grains in cultivars under test. Soil analysis immediately following solarization revealed that essential elements were readily available in simpler forms, which may have increased pest resistance and reduced stalk breakage.     

Pathogenicity of<Emphasis Type="Underline">Fusarium</Emphasis><Emphasis Type="Underline">SPP</Emphasis>. contributing to the stalk rot of Maize in Poland

During 1985–1989 a stalk rot of early maturity hybrids of maize was studied in Radzikow (Central Poland). It was found thatFusarium species were dominant on plants with stalk rot symptoms. Spectrum ofFusarium spp. had changed within the years. The most frequently isolated were:F.subglutinans,F.culmorum andF.crookwellense. When the disease developed early in the seasonF.graminearum was also present.Predominant species were examined for their pathogenicity according to the modified method of Molot, Simone (1967). Isolates ofF.graminearum andF.culmorum were found to be strong pathogens,F.crookwellense andF.subglutinans — moderate andF.oxysporum andF.eguiseti were the weak ones     

Fungi Associated with Foot Rots on Winter Wheat in Northwest Italy

In a 3-year survey on bread and durum wheat grown in Northwest Italy, brown root rot was the most important disease of the basal part of culm. Year, wheat-growing area, cultivar and their interactions significantly influenced brown root rot incidence. The most important fungal species isolated from lower stems with browning were Microdochium nivale, Drechslera sorokiniana, Fusarium avenaceum, F. graminearum, and F. culmoniliforme, and Pythium spp. were frequently isolated. F., crookwellense was also isolated. Sharp eyespot was a frequent disease; take-all and eyespot occurred only occasionally.     

Fusarium species and their mycotoxins in infected corn in Italy

Surveys of corn (infected plants and commercial kernels) forFusarium species and their mycotoxins were carried out on samples collected all over Italy and from some European and mediterranean countries.Investigations on samples of corn stalk and ear rot standing in the field, mainly collected in southern Italy, proved to be contaminated with zearalenone (ZON), zearalenols (ZOL), and deoxynivalenol (DON). TheFusarium species most frequently isolated, and their recorded toxigenic capability (in parentheses), were:F. moniliforme;F. culmorum (ZON, ZOL, DON, 3AcDON);F. equiseti (ZON, ZOL); andF. proliferatum (MF). Along with these species,F. graminearum group 2 (ZON, DON and/or 3AcDON or 15AcDON);F. chlamydosporum;F. acuminatum (type-A trichothecene derivatives); andF. semitectum were often found to be associated.F. heterosporum (ZON, ZOL);F. solani;F. crookwellense (ZON, ZOL, FUS, NIV);F. oxysporum (MF);F. avenaceum (MF);F. sporotrichioides (T-2 toxin and derivatives); andF. poae (DAS, MAS) were occasionally isolated.     

The inability of Fusarium species extracts to induce dieback in pines

Cultures of F. moniliforme var. subglutinans, F. moniliforme, F. lateritium, F. equiseti, F. semitectum and F. solani from pine and F. moniliforme and F. graminearum from southern U.S. corn were grown on rice and corn, extracted, and checked for toxicity in mice, chicken embryos, and pine seedlings, and for mutagenicity by the Ames test. While extracts from both fungal groups contained toxins, none of the extracts induced dieback in pine seedlings. Almost all of the cultures isolated from corn in contrast to those from pine, were mutagenic. Thin-layer chromatography did not detect T-2 toxin, moniliformin, or vomitoxin, indicating that these toxins do not elicit dieback symptoms in pine.     

Species-specific PCR Assays for the Fungal Pathogens Fusarium moniliforme and Fusarium subglutinans and their Application to Diagnose Maize Ear Rot Disease

Fusarium moniliforme Sheldon (syn. F. verticillioides (Sacc.) Nirenberg) and F. subglutinans (Wollenweber & Reinking) Nelson Toussoun & Marasas comb. nov., two anamorphs of the so-called‘Gibberella fujikuroi species complex', are important maize pathogens. Together with F. proliferatum, F. culmorum, and F. graminearum (teleomorph: Gibberella zeae) they are involved in the stalk rot and ear rot disease of maize. All species produce secondary metabolites (mycotoxins) which are a potential health hazard for humans and animals that consume maize and maize products frequently. In this study the development of polymerase chain reaction (PCR) assays for an easy and sensitive identification of G. fujikuroi anamorphs in maize kernels are described. The primer pairs are based on sequences of randomly amplified polymorphic DNA (RAPD) fragments and are specific for F. moniliforme and F. subglutinans respectively. The PCR assays are independent of the high phenotypic variability of traits which may complicate classification by morphological characters. They detect approximately 100 to 200 fungal genomes in the presence of an excess of maize DNA. For the analysis of infected maize kernels a rapid and easy DNA extraction was used which does not introduce inhibitory substances into the PCR. Hence the assays enable an early identification and detection of the two pathogens in host tissue by plant breeders and plant health inspection services. The assays were successfully applied to identify field isolates from Poland and to detect the pathogens in maize ears of various hybrids in Germany.     

Effect of moisture stress, plant population density and pathogen inoculation on charcoal stalk rot of sorghum

The effects of moisture deficit stress, plant population density and pathogen inoculation technique on charcoal stalk rot in the sorghum hybrid CSH 6 were studied in the 1980–81 and 1981–82 post-rainy seasons at three locations in India. Incidence and severity of charcoal rot caused by Macrophomina phaseolina were compared in three plant population densities, subjected to different moisture stress regimes created by withholding irrigation at various plant growth stages. Natural infections were compared to artificial inoculation with M. phaseolina. Combinations of moisture stress, plant population and inoculation treatments were compared to identify the combination most likely to develop maximum disease. Lodging, the first external symptom of charcoal rot, was significantly correlated with other disease symptoms used to measure charcoal rot, such as soft stalk, number of nodes crossed by M. phaseolina infection, root damage and plant senescence. In both seasons the highest incidence of lodging occurred when moisture stress was induced at the 'flag leaf visible in the whorl' growth stage. The greatest incidence of the disease was recorded in the highest plant population (266 700 plant ha^-) at all three locations. No significant differences were found between artificially and naturally inoculated treatments. The maximum number of lodged plants was found at a density of 266 700 plants ha^-1 when moisture stress was induced at the 'flag leaf visible in the whorl' growth stage.     

Covariation between line and testcross performance for reduced mycotoxin concentrations in European maize after silk channel inoculation of two <Emphasis Type="Italic">Fusarium</Emphasis> species

Fusarium spp. in maize can contaminate grain with mycotoxins harmful to humans and animals. Breeding and growing resistant varieties is one alternative to reduce contamination by mycotoxins. Little is known about the population parameters relevant to resistance breeding. The objectives of this study were to draw conclusions on breeding of reduced mycotoxin concentrations of deoxynivalenol, zearalenone and fumonisins, and resistance to ear rot after silk channel inoculation with F. graminearum or F. verticillioides, respectively. For that, variation and covariation of line and testcross performance and correlations between both species and between mycotoxin concentrations and ear rot resistance were calculated. Means of ear rot after infection with F. graminearum were higher than with F. verticillioides. Moderate phenotypic correlations (r = 0.46–0.65) between resistances to both Fusarium spp. implicate the need of separate testing. Analyses of variance revealed significant (P < 0.01) differences among lines in line and testcross performance for 30–60 entries per maturity group. Multi-environmental trials for accurate selection are necessary due to significant (P < 0.1) genotype × environment interactions. High genotypic correlations between ear rots and mycotoxins (r ≥ 0.90), and similar heritabilities of both traits, revealed the effectiveness of indirect selection for mycotoxin concentrations based on ear rot rating after inoculation. Moderate genotypic correlations between line and testcross performance were found (r = 0.64–0.83). The use of one moderately to highly susceptible tester is sufficient since genotypic correlations between testcrosses of different testers were high (r = 0.80–0.94). Indirect selection for testcross performance based on line performance is less effective than selection based on mycotoxin concentrations. Consequently, selection for resistance to ear rot and mycotoxin accumulation should be started among testcrosses tested first for general combining ability based on ear rot data in parallel with a negative selection for line per se performance.     

Emetic material associated with Fusarium species in cereal grains and artificial media

Substances which cause emesis in pigeons were extracted from corn (Zea mays) artificially inoculated with Fusarium graminearum and from liquid culture medium inoculated with F. moniliforme, F. roseum, F. poae, F. culmorum, and F. nivale. Emetic preparations were obtained also from infected wheat (Triticum aestivum L. em. Thell), (Hordeum vulgare L. em. Lam), and durum (Triticum durum Desf). Partial purification resulted from chromatography with columns of cellulose and DEAE cellulose and with thin layers of silica gel. Two active materials were obtained from liquid culture of F. moniliforme but only one from infected cereals. Emetic preparations from F. moniliforme and infected cereals contained a polypeptide as a minor component. Ultraviolet and infrared spectrums, elemental analyses, refractive indices, and amino acid composition of the emetic from corn and one of the emeties from liquid culture of F. moniliforme were similar but not identical. Attempts to crystalline these emetics and to characterize them were unsuccessful.     

Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa⁺), siderophore (Sid⁺), HCN (HCN⁺) and fluorescent pigments (Flu⁺) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid⁺), antibiotics (Afa⁺) and antifungal volatiles (Afv⁺), MRF exhibited the production of antifungal antibiotics (Afa⁺) and siderophores (Sid⁺). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lac Z induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lac Z mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lac Z marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

Production of zearalenone,T-2 toxin,and deoxynivalenol by Fusarium spp. isolated from plant materials grown in North Carolina

Fusarium spp. isolated from plant materials grown in the hot, humid climate of North Carolina were tested for production of mycotoxins. Isolates of F. acuminatum, F. graminearum, F. moniliforme, F. oxysporum, and F. solani produced zearalenone while isolates of F. equiseti and F. graminearum produced T-2 toxin and deoxynivalenol, respectively. This is the first report of zearalenone production by F. solani. The toxins were identified by capillary gas chromatography-mass spectrometry. These findings suggest that there are toxigenic strains of Fusarium indigenous to the warmer regions of the USA and that fasariotoxicoses of animals in this region are not necessarily the result of importing toxic grains from the cooler, upper midwestern USA.Paper No. 8953 of the Journal Series of North Carolina Agricultural Research Service, Raleigh, North Carolina. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the product named nor criticism of similar ones not mentioned.     

Use of molecular markers for monitoring fungi involved in stalk rot of corn

Summary Separate genes conferring antibiotic drag resistance have been inserted into Fusarium graminearum and Fusarium moniliforme. These organisms are associated with stalk rot of corn, a disease of uncertain cause. Antibiotic resistant fungi were obtained by developing a gene transfer system using whole cells as recipients for DNA. Hygromycin B and benomyl-resistant colonies were isolated by treating fungal tissue with lithium acetate and adding plasmid vectors containing the respective genes which give drug resistance. The DNA was stably integrated into the fungal chromosome. Following plant inoculation, disease symptoms developed and the isolates were recovered on selective medium. In each case, these fungi retained the transformed phenotype, although extensive rearrangements and/or deletions occurred. Specific molecular tagging allows detailed studies of this interaction and should be of general use in situations involving complex multiple pathogen diseases.     

A major QTL for resistance to Gibberella stalk rot in maize

Fusarium graminearum Schwabe, the conidial form of Gibberella zeae, is the causal fungal pathogen responsible for Gibberella stalk rot of maize. Using a BC₁F₁ backcross mapping population derived from a cross between ‘1145’ (donor parent, completely resistant) and ‘Y331’ (recurrent parent, highly susceptible), two quantitative trait loci (QTLs), qRfg1 and qRfg2, conferring resistance to Gibberella stalk rot have been detected. The major QTL qRfg1 was further confirmed in the double haploid, F₂, BC₂F₁, and BC₃F₁ populations. Within a qRfg1 confidence interval, single/low-copy bacterial artificial chromosome sequences, anchored expressed sequence tags, and insertion/deletion polymorphisms, were exploited to develop 59 markers to saturate the qRfg1 region. A step by step narrowing-down strategy was adopted to pursue fine mapping of the qRfg1 locus. Recombinants within the qRfg1 region, screened from each backcross generation, were backcrossed to ‘Y331’ to produce the next backcross progenies. These progenies were individually genotyped and evaluated for resistance to Gibberella stalk rot. Significant (or no significant) difference in resistance reactions between homozygous and heterozygous genotypes in backcross progeny suggested presence (or absence) of qRfg1 in ‘1145’ donor fragments. The phenotypes were compared to sizes of donor fragments among recombinants to delimit the qRfg1 region. Sequential fine mapping of BC₄F₁ to BC₆F₁ generations enabled us to progressively refine the qRfg1 locus to a ~500-kb interval flanked by the markers SSR334 and SSR58. Meanwhile, resistance of qRfg1 to Gibberella stalk rot was also investigated in BC₃F₁ to BC₆F₁ generations. Once introgressed into the ‘Y331’ genome, the qRfg1 locus could steadily enhance the frequency of resistant plants by 32–43%. Hence, the qRfg1 locus was capable of improving maize resistance to Gibberella stalk rot.