Acremonium Zeae Research Articles

Fungal Mycoflora Associated with Busseola fusca Frass in Maize Plants

Busseola fusca and Fusarium verticillioides are considered the most important pest and pathogen, respectively, of maize in South Africa. Busseola fusca tunnels into stems and ears of maize plants, whereas F. verticillioides causes Fusarium ear rot and deposits fumonisins in maize kernels. Frass (excreta) deposited by B. fusca larvae in maize stems and ears during feeding could potentially be contaminated with pathogens such as F. verticillioides, thereby indirectly and passively contributing to plant diseases. The mycoflora present in frass of B. fusca larvae was thus investigated in this study, and the dissemination of fumonisin-producing Fusarium spp. by B. fusca larvae in maize stems determined. Busseola fusca frass was collected from plants with visible insect damage in three maize-growing districts in South Africa. The mycoflora in the frass was isolated and identified morphologically to genus level, followed by the sequencing of target genes for species identification. Species of Acremonium, Aspergillus, Fusarium, Mucor, Rhizopus and Talaromyces were associated with the B. fusca frass. The role of B. fusca larvae in disseminating fumonisin-producing Fusarium spp. was then determined in greenhouse and field trials. Maize whorls were inoculated with F. verticillioides MRC826 spores 4 weeks after plant emergence, and infested with B. fusca larvae 2 days later. The stems were split open after 3 weeks and frass collected from feeding channels, thereafter, target DNA of fumonisin-producing Fusarium spp. was quantified using real-time PCR. Target DNA of fumonisin-producing Fusarium spp. was significantly higher in frass collected from greenhouse plants inoculated with F. verticillioides than in frass collected from the uninoculated control, indicating that the inoculation was successful in the absence of soil-borne inoculum. Nevertheless, the field trial showed no significant differences in target DNA in frass from inoculated and non-inoculated plants. This is possibly due to natural F. verticillioides infection of maize plants in the field. This study further indicated that B. fusca frass also contained maize pathogens such as Aspergillus spp. and Fusarium spp., which could cause ear rot diseases. The occurrence of Acremonium zeae in frass has potential implications for the biological control of F. verticillioides, as the fungus produces pyrrocidines A and B antibiotics which are known to be antagonistic to F. verticillioides. The occurrence of Aspergillus niger in frass requires further investigation in order to clarify its role in fumonisin contamination of maize in South Africa.

ChemInform Abstract: Efficient Total Synthesis of (S)‐Dihydroresorcylide, a Bioactive Twelve‐Membered Macrolide.

AbstractA total synthetic approach towards the title compound (VII), a natural compound from Acremonium zeae with interesting phytotoxic activity, is presented from orcinol monohydrate and involves 9 steps.

Direct ethanol production from glucose, xylose and sugarcane bagasse by the corn endophytic fungi Fusarium verticillioides and Acremonium zeae

Production of ethanol with two corn endophytic fungi, Fusarium verticillioides and Acremonium zeae, was studied. The yield of ethanol from glucose, xylose and a mixture of both sugars were 0.47, 0.46 and 0.50g/g ethanol/sugar for F. verticillioides and 0.37, 0.39 and 0.48g/g ethanol/sugar for A. zeae. Both fungi were able to co-ferment glucose and xylose. Ethanol production from 40g/L of pre-treated sugarcane bagasse was 4.6 and 3.9g/L for F. verticillioides and A. zeae, respectively, yielding 0.31g/g of ethanol per consumed sugar. Both fungi studied were capable of co-fermenting glucose and xylose at high yields. Moreover, they were able to produce ethanol directly from lignocellulosic biomass, demonstrating to be suitable microorganisms for consolidated bioprocessing.

Efecto tóxico de Acremonium zeae en pollos de engorda en iniciación

El objetivo de este trabajo fue determinarsi Acremonium zeae afecta el desarrollo de pollos deengorde. En el Colegio de Postgraduados, Campus Montecillo,en el Estado de Mexico de agosto a diciembre de 2010,se realizo un experimento con pollos recien nacidos bajoun diseno experimental completamente al azar con cincodietas (tratamientos) y cinco repeticiones, cada una constode cinco pollos. Los animales fueron alimentados durantetres semanas con cada una de las dietas, en las que se adicionaronporcentajes de avena contaminada con A. zeae ensustitucion del maiz. Los tratamientos fueron: T1, 0% avenacontaminada: 100% de maiz; T2, 25% avena contaminada:75% de maiz; T3, 50% avena contaminada: 50% de maiz;T4, 75% avena contaminada: 25% de maiz; y, T5, 100%avena contaminada: 0% de maiz. Durante tres semanas cadasiete dias se registro el incremento en peso de los pollos yel consumo de alimento. Hubo efecto negativo en el peso apartir de la segunda semana de alimentacion, con una composicionde dieta elaborada con un minimo de 50% de avenacontaminada con el hongo.

Bacterial and fungal endophytes associated with grains and roots of maize

The study was carried out to determine the microbes of maize grains sourced from five markets in Akungba and Ikare-Akoko, Ondo State, Nigeria. Bacterial and fungal microbes from roots of two maize cultivars DMR-LSR-Y and TZMSR-W were also investigated using the pour plate method. Results showed that grains from Oja Oba had the highest bacterial population of 4.8 × 105 cfu/g, while, the highest fungal load of 4.1 × 103 cfu/g was obtained from Osele market in Ikare. The two maize cultivars showed generally a low fungal count compared to their bacterial counterparts with 1.1 × 105 cfu/g root for a cultivar DMR-LSR-Y and 0.2 × 105 cfu/g root for TZMSR-W. The dry white maize grains showed generally low bacterial and fungal colonizations of 0.2 × 105 and 0.3 × 103 cfu/g respectively when compared to dry and fresh yellow types. Eleven bacteria genera and eight fungal species were isolated and identified from the roots and grains of maize. These includeCellulomonas, Bacillus, Pseudomonas, Staphylococcus, Micrococcus, Pediococcus, Microbacterium, Azospirillum, Kurtia, and Enterobacter, Acremonium zeae, Alternaria alternata, Aspergillus flavus, Aspergillus niger, Colletotrichum graminicola, Fusarium verticillioides, Saccharomyces cerevisiae, and Trichoderma koningii. The study was important in bioprospecting for biological activities and plant growth enhancers. Key words: Bacterial population, fungal population, endophytes, grains, roots, maize.

Purification and characterization of arabinofuranosidase from the corn endophyte Acremonium zeae

Acremonium zeae, one of the most prevalent fungal colonists of preharvest corn, possesses a suite of hemicellulolytic activities including xylanase, xylosidase, and arabinofuranosidase. Two enzymes with arabinofuranosidase activity were purified from cell-free culture supernatants of A. zeae grown on oat spelt xylan. A 47 kDa enzyme (AF47) was optimally active at 37 °C and pH 6.0, and had a specific activity for 4-nitrophenyl-α-L-arabinofuranoside (4NPA) of 6.2 U/mg. A 30 kDa enzyme (AF30) was optimally active at 50 °C and pH 4.5, and had a specific activity for 4NPA of 12.4 U/mg. AF47 hydrolyzed 4-nitrophenyl-β-D-xylopyranoside, 4-nitrophenyl-β-D-glucopyranoside, and 4-nitrophenyl-β-D-cellobioside, as well as producing reducing sugars from corn fiber, wheat, and oat spelt arabinoxylan. AF30 had little detectable activity on the 4-nitrophenyl substrates, except for 4NPA, but activity on arabinoxylans from corn fiber, wheat, and oat spelt was at least 7-fold higher than AF47, with specific activities of 109, 358, and 153 U/mg, respectively. A combination of the two enzymes released 61 and 88% of the total arabinose from corn fiber and wheat arabinoxylans. The arabinofuranosidases produced by A. zeae may have industrial application for the enzymatic hydrolysis of recalcitrant lignocellulosic feedstocks such as corn fiber and wheat straw.

Cellulases and Hemicellulases from Endophytic Acremonium Species and Its Application on Sugarcane Bagasse Hydrolysis

The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing L -arabinose, D -xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using D -xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose.

Extracellular Hemicellulolytic Enzymes from the Maize Endophyte Acremonium zeae

Microorganisms that colonize plants require a number of hydrolytic enzymes to help degrade the cell wall. The maize endophyte Acremonium zeae was surveyed for production of extracellular enzymes that hydrolyze cellulose and hemicellulose. The most prominent enzyme activity in cell-free culture medium from A. zeae NRRL 6415 was xylanase, with a specific activity of 60 U/mg from cultures grown on crude corn fiber. Zymogram analysis following SDS-PAGE indicated six functional xylanase polypeptides of the following masses: 51, 44, 34, 29, 23, and 20 kDa. Xylosidase (0.39 U/mg), arabinofuranosidase (1.2 U/mg), endoglucanase (2.3 U/mg), cellobiohydrolase (1.3 U/mg), and beta-glucosidase (0.85 U/mg) activities were also detected. Although apparently possessing a full complement of hemicellulolytic activities, cell-free culture supernatants prepared from A. zeae required an exogenously added xylosidase to release more than 90% of the xylose and 80% of the arabinose from corn cob and wheat arabinoxylans. The hydrolytic enzymes from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars.

Antimicrobial Activity of Pyrrocidines from Acremonium zeae Against Endophytes and Pathogens of Maize

Acremonium zeae produces pyrrocidines A and B, which are polyketide-amino acid-derived antibiotics, and is recognized as a seedborne protective endophyte of maize which augments host defenses against microbial pathogens causing seedling blights and stalk rots. Pyrrocidine A displayed significant in vitro activity against Aspergillus flavus and Fusarium verticillioides in assays performed using conidia as inoculum, with pyrrocidine A being more active than B. In equivalent assays performed with conidia or hyphal cells as inoculum, pyrrocidine A revealed potent activity against major stalk and ear rot pathogens of maize, including F. graminearum, Nigrospora oryzae, Stenocarpella (Diplodia) maydis, and Rhizoctonia zeae. Pyrrocidine A displayed significant activity against seed-rotting saprophytes A. flavus and Eupenicillium ochrosalmoneum, as well as seed-infecting colonists of the phylloplane Alternaria alternata, Cladosporium cladosporioides, and Curvularia lunata, which produces a damaging leaf spot disease. Protective endophytes, including mycoparasites which grow asymptomatically within healthy maize tissues, show little sensitivity to pyrrocidines. Pyrrocidine A also exhibited potent activity against Clavibacter michiganense subsp. nebraskense, causal agent of Goss's bacterial wilt of maize, and Bacillus mojaviense and Pseudomonas fluorescens, maize endophytes applied as biocontrol agents, but were ineffective against the wilt-producing bacterium Pantoea stewartii.

Occurrence of pyrrocidine and dihydroresorcylide production among Acremonium zeae populations from maize grown in different regions

Acremonium zeae is recognized as a protective endophyte of maize (Zea mays). Thus, it is a potential confounding variable in maize variety trials for resistance to pathogenic microbes and their mycotoxins. This fungus grows systemically in maize and produces pyrrocidines A and B, polyketide – amino acid derived antibiotics exhibiting potent in vitro activity against major stalk- and ear-rot pathogens of maize. An evaluation of a number of A. zeae isolates accessioned by the USDA Agricultural Research Service Culture Collection (NRRL) and CBS Culture Collections from 1969 to 1992 revealed that pyrrocidines were produced by 5 of 11 isolates from maize grown in regions sharing a milder temperate climate with cold winters (e.g., Illinois, Nebraska, and Germany) and by 10 of 12 isolates from maize grown in warmer regions (e.g., North Carolina and Georgia, USA; Iran; and India). Cultures of A. zeae representing 13 populations isolated from maize seeds harvested from different regions and years were grown on potato dextrose agar in Petri dishes. Excised portions of the developing colony and underlying agar were extracted and analyzed by liquid chromatography – atmospheric pressure chemical ionization interface – mass spectrometry. Among 52 isolates representing four A. zeae populations from regions associated with drought and temperature stress (e.g., Arizona, California, and Texas) 49 isolates (94%) produced pyrrocidine A in amounts ranging from 1 to 248 ng (mean 29 ng), and 51 isolates (98%) produced pyrrocidine B in amounts ranging from 2 to 440 ng (mean 137 ng). In contrast, among 102 isolates representing nine A. zeae populations from the midwestern corn belt (e.g., Illinois, Indiana, Iowa, Kentucky, Michigan, and Nebraska), only one isolate produced pyrrocidine A (280 ng) and pyrrocidine B (411 ng). During the population study, a new secondary metabolite of A. zeae, dihydroresorcylide, was discovered, and its distribution among the population is described. Clonal populations of A. zeae endophytes differing in their ability to produce pyrrocidines may be distributed with seed of maize cultivars grown in commercial plantings. Key words: Acremonium zeae, blackened bundles, cerebroside, dihydroresorcylide, disease suppression, endophyte, maize, polyketide antibiotics, pyrrocidines, stress damage, seedborne. Acremonium zeae est reconnu en tant que champignon endophyte protecteur du maïs. Il s’agit par conséquent d’un facteur confusionnel potentiel dans les essais de variété effectués sur le maïs afin d’en évaluer la résistance aux microbes pathogènes et à leurs mycotoxines. A. zeae pousse systématiquement sur le maïs et produit des pyrrocidines A et B, c’est-à-dire des antibiotiques polykétides dérivés d’acides aminés qui affichent une forte activité in vitro contre les principaux agents pathogènes causant la fusariose de l’épi et la pourriture des tiges chez le maïs. Une évaluation d’un nombre d’isolats d’ A. zeae, répertoriés, de 1969 à 1992, par l’Agricultural Research Service Culture Collection NRRL et le CBS Culture Collection a démontré que 5 des 11 isolats obtenus de plants de maïs cultivés dans des régions qui connaissent des étés doux et des hivers froids (p. ex., Illinois, Nebraska, Allemagne) produisaient des pyrrocidines, de même que 10 des 12 isolats cultivés dans des régions plus chaudes (p. ex., Caroline du Nord et Géorgie, États-Unis; Iran; Inde). Afin de les faire croître, des cultures d’ A. zeae, représentant 13 populations isolées à partir de semences de maïs récoltées dans diverses régions et au cours d’années différentes, ont été placées dans des boîtes de Pétri sur de la gélose dextrosée à la pomme de terre. Des portions isolées de colonies et de gélose sous-jacente ont été extraites et analysées par LC-APCI-MS. Parmi les 52 isolats représentant 4 populations d’ A. zeae provenant de régions associées aux stress causés par la sécheresse et la température (p. ex., Arizona, Californie, Texas), 49 isolats (94 %) ont produit de la pyrrocidine A en quantité variant de 1 à 248 ng (moyenne de 29 ng) et 51 isolats (98 %) ont produit de la pyrrocidine B dont les quantités variaient de 2 à 440 ng (moyenne de 137 ng). Par ailleurs, parmi les 102 isolats représentant 9 populations d’ A. zeae issues de la ceinture de maïs des États du Midwest (p. ex., Illinois, Indiana, Iowa, Kentucky, Michigan, Nebraska), un seul isolat a produit de la pyrrocidine A (280 ng) et de la pyrrocidine B (411 ng). Au cours de l’étude de populations, un nouveau métabolite secondaire d’ A. zeae, le dihydroresorcylide, a été découvert et l’on décrit sa distribution dans la population. Des semences de cultivars de maïs cultivées commercialement peuvent contenir des populations clonales d’endophytes d’ A. zeae dont la capacité à produire des pyrrocidines est variable. Mots-clés : Acremonium zeae, maladie des faisceaux noirs, cérébroside, dihydroresorcylide, suppression des maladies, endophyte, antibiotiques polykétides, pyrrocidines, dommages dus au stress, transmis par semence.

Acremonium zeae, a Protective Endophyte of Maize, Produces Dihydroresorcylide and 7-Hydroxydihydroresorcylides

Acremonium zeae has been characterized as a protective endophyte of maize and displays antifungal activity against other fungi. Pyrrocidines A and B were discovered to be the metabolites accounting for this activity. During a population survey of A. zeae isolates from maize seeds produced in nine states to determine their ability to produce pyrrocidines, another metabolite of A. zeae, unrelated to the pyrrocidines, was found to have widespread occurrence (105 of 154 isolates) and to be produced in amounts comparable to the pyrrocidines. Further chemical studies of fermentation extracts of an A. zeae isolate (NRRL 45893) from maize led to the identification of a new compound, dihydroresorcylide, the saturated analogue of cis-resorcylide. Also identified were the two diastereomers of 7-hydroxydihydroresorcylide. Dihydroresorcylide and pyrrocidines A and B were detected by LC-APCI-MS in symptomatic maize kernels from ears that were wound-inoculated in the milk stage with A. zeae NRRL 34559.

Host-synthesized secondary compounds influence the in vitro interactions between fungal endophytes of maize.

Maize produces a suite of allelopathic secondary metabolites, the benzoxazinoids. 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and 2,4-dihydroxy-2H-1,4-benzoxazin-3-one reside as glucosides in plant tissue and spontaneously degrade to 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA) upon plant cell disruption. Several maize-associated fungi in the genus Fusarium can metabolize MBOA and BOA. BOA tolerance levels in 10 species of Fusarium and in the maize endophytes Nigrospora oryzae, Acremonium zeae, and Periconia macrospinosa were characterized. BOA tolerance ranged from 0.25 to 1.10 mg/ml among species. The influence of substrate alteration by one species on the subsequent growth of another species was assessed in the presence and absence of BOA. The colony area of the secondary colonizer in heterospecific interactions was compared to that in autospecific interactions (one isolate follows itself). In the presence of BOA, four of six secondary colonizers had greater growth (facilitation) when primary colonizers had higher BOA tolerance than the secondary colonizer. When the primary colonizer had lower tolerance than the secondary, three of six secondary colonizers were inhibited (competition) and three not significantly affected. In BOA-free medium, the number of isolates that were facilitated or inhibited was the same regardless of the tolerance level of the primary colonizer. Two of six secondary colonizers were facilitated, two inhibited, and two not significantly affected. This study provides some support for facilitation in stressful conditions under the Menge-Sutherland model. The results are not consistent with the corresponding prediction of competition in the absence of stress. The hypothesis drawn from these data is that in the presence of a toxin, fungal species that detoxify their substrate can enhance the colonization rate of less tolerant fungi.

A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides

The maize endophyte Acremonium zeae is antagonistic to kernel rotting and mycotoxin producing fungi Aspergillus flavus and Fusarium verticillioides in cultural tests for antagonism, and interferes with A. flavus infection and aflatoxin contamination of preharvest maize kernels. Chemical studies of an organic extract from maize kernel fermentations of Acremonium zeae (NRRL 13540), which displayed significant antifungal activity against Aspergillus flavus and F. verticillioides, revealed that the metabolites accounting for this activity were two newly reported antibiotics pyrrocidines A and B. Pyrrocidines were detected in fermentation extracts for 12 NRRL cultures of Acremonium zeae isolated from maize kernels harvested in Illinois (4/4 cultures), North Carolina (5/5), Georgia (1/2) and unrecorded locations within the USA (2/2). Pyrrocidine B was detected by LCMSMS in whole symptomatic maize kernels removed at harvest from ears of a commercial hybrid that were wound-inoculated in the milk stage with A. zeae (NRRL 13540) or (NRRL 13541). The pyrrocidines were first reported from the fermentation broth of an unidentified filamentous fungus LL-Cyan426, isolated from a mixed Douglas Fir hardwood forest on Crane Island Preserve, Washington, in 1993. Pyrrocidine A exhibited potent activity against most Gram-positive bacteria, including drug-resistant strains, and was also active against the yeast Candida albicans. In an evaluation of cultural antagonism between 13 isolates of A. zeae in pairings with A. flavus (NRRL 6541) and F. verticillioides (NRRL 25457), A. zeae (NRRL 6415) and (NRRL 34556) produced the strongest reaction, inhibiting both organisms at a distance while continuing to grow through the resulting clear zone at an unchanged rate. Maximum colony diameters for A. zeae (NRRL 6415) and (NRRL 13540), on potato dextrose agar after 14 d, were attained within the range of 25-30 degrees C, with less growth recorded at 15 degrees and 37.5 degrees and no growth at 5 degrees. Potential interactions between A. zeae and other maize endophytes are considered and the significance of these interactions relative to the aflatoxin and fumonisin contamination of preharvest maize is presented. This is the first report of natural products from Acremonium zeae.

Mycotoxins and toxigenic fungi in insect-damaged maize harvested during 1983 in far North Queensland

A survey for mycotoxins and toxigenic fungi in maize (Zea mays) grown during 1983 in Far North Queensland is reported. Zearalenone was detected in 148 of the 174 samples examined. Low concentrations of aflatoxins were detected in three of the samples. Ochratoxin A, sterigmatocystin, T-2 toxin and deoxynivalenol were not detected. The 1983 season was unusual in that plants were stressed from low rainfall in the early stage of growth. Insect damage, mainly by ear worms (Heliothis sp.), was severe. Despite this, there was no significant increase in zearalenone concentrations compared to a previous survey in 1982, when insect damage was minimal. Ten samples were used for fungal isolations. Fusarium moniliforme was the most frequently isolated fungus, and its isolation frequency increased in visibly damaged kernels. F. graminearum was most frequently isolated from kernels with a purple discoloration. The correlation between the isolation frequency of F. graminearum and zearalenone concentrations indicated that F. graminearum produced most of the zearalenone in the samples. Other fungi isolated included F. subglutinans, F. oxysporum, Aspergillus jlavus, A. niger, Penicillium sp., Trichoderma sp., Nigrospora sp., Curvularia sp., Drechslera sp., Acremonium zeae and Rhizoctonia sp.