Aspergillus oryzae nrtA affects kojic acid production

We analyzed the role of the nitrate transporter-encoding gene (nrtA) of Aspergillus oryzae by gene disruption. Southern hybridization analysis indicated that homologous recombination occurred at the resident nrtA locus. Real-time PCR showed that the nrtA gene was strongly inducible by NaNO₃. The nrtA disruptant did not exhibit normal growth when nitrate was available as the sole nitrogen source. These results indicate that NrtA is essential for nitrate uptake in A. oryzae. Kojic acid (KA) production was inhibited by the addition of a small amount of sodium nitrate. The nrtA-disrupted strain was deficient in the uptake of nitrate. As a result, KA production in this strain was not considerably affected by the presence of nitrate.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Trichoderma theobromicola and T. paucisporum: two new species isolated from cacao in South America

Trichoderma theobromicola and T. paucisporum spp. nov. are described. Trichoderma theobromicola was isolated as an endophyte from the trunk of a healthy cacao tree (Theobroma cacao, Malvaceae) in Amazonian Peru; it sporulates profusely on common mycological media. Trichoderma paucisporum is represented by two cultures that were obtained in Ecuador from cacao pods partially infected with frosty pod rot, Moniliophthora roreri; it sporulates sporadically and most cultures remain sterile on common media and autoclaved rice. It sporulates more reliably on synthetic low-nutrient agar (SNA) but produces few conidia. Trichoderma theobromicola was reintroduced into cacao seedlings through shoot inoculation and was recovered from stems but not from leaves, indicating that it is an endophytic species. Both produced a volatile/diffusable antibiotic that inhibited development of M. roreri in vitro and on-pod trials. Neither species demonstrated significant direct in vitro mycoparasitic activity against M. roreri.     

Verticillium wilt of cacao in Uganda: symptoms and establishment of pathogenicity

Pathogenicity tests have shown that Verticillium dahliae Kleb. causes a vascular wilt disease of cacao (Theobroma cacao L.) in Uganda. Isolates of V. dahliae from cacao, okra (Abelmoschus esculentus Medik.), and cotton (Gossypium hirsutum L.) induced similiar symptoms on artificially inoculated cacao seedlings. External symptoms of the disease include acute wilting, foliar chlorosis and abscission, stunting of roots and shoots, and production of suckers at lower nodes of severely affected plants. The internal symptoms are vascular discoloration, tylosis, and presence of gum deposits and mycelium of V. dahliae in xylem vessels.     

Spread and persistence of a rugulosin-producing endophyte in Picea glauca seedlings

We have studied Picea glauca (white spruce) endophyte colonization and its affect on the growth of Choristoneura fumiferana (spruce budworm). Here we examine the spread and persistence of a rugulosin-producing endophyte and rugulosin in needles from trees maintained in the nursery, as well as in trees planted in a test field site. Additionally, we report toxicity of rugulosin against three P. glauca needle herbivores: C. fumiferana, Lambdina fiscellaria (hemlock looper) and Zeiraphera canadensis (spruce budmoth). Reduction in body weight for both the C. fumiferana and L. fiscellaria were observed at 25 and 50 μm, respectively, and head capsules were reduced at 100 and 150 μm. Z. canadensis larvae did not perform as well in tests due to an Aspergillus fumigatus infection, but were shown to be lighter when tested with 100 and 150 μm compared with controls. The endophyte and its toxin were shown to spread throughout the nursery-grown seedlings. After 3.5 and 4.5 y post-inoculation (one and two years in the test site), the inoculated endophyte and its toxin had remained present with an average rugulosin concentration of 1 μg g⁻¹.     

Microbiology of the root region of coconut and cacao under mixed cropping

Summary Mixed cropping of cacao in coconut plantations improved the microbial activity in the rhizosphere of coconut which may be attributed to an increase in organic matter content of soil due to periodic shedding of cacao leaves. When compared to coconut cultivation without cacao, mixed cropping of coconut with cacao appeared to stimulate the population of bacteria and fungi in the rhizosphere of coconut, including the nitrogen fixing and phosphate solubilising bacteria. The occurrence of indole acetic acid producing Escherichia sp. on the root surface of coconut and the appearance of gibberellin(like-substance) producing Aspergillus flavus and A. fumigatus in the rhizosphere were other salient observations made in a pioneering study on the microbiology of the root region of plantation crops under mixed cropping. re]19750702     

Effects of Colonization of a Bacterial Endophyte,Azospirillum sp. B510, on Disease Resistance in Rice

Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with B510 exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In the rice plants, neither salicylic acid (SA) accumulation nor expression of pathogenesis-related (PR) genes was induced by interaction with this bacterium, except for slight induction of PBZ1. These results indicate the possibility that strain B510 is able to induce disease resistance in rice by activating a novel type of resistance mechanism independent of SA-mediated defense signaling.     

First record of Aspergillus oryzae (Eurotiales: Trichocomaceae) as an entomopathogenic fungus of the locust,Locusta migratoria (Orthoptera: Acrididae)

A novel entomopathogenic fungus of Locusta migratoria was identified as Aspergillus oryzae using a comparative sequence analysis of the internal transcribed spacer regions, aflatoxin B₁ detection and morphological analysis. The fungus isolated from a dead locust collected in northwestern China was found to be pathogenic to the insect. Phylogenetic experiments revealed a 99% similarity between the fungus and those of three species, A. oryzae, Aspergillus flavus and Aspergillus parvisclerotigenus which are in the same branch of the Flavi section of the genus Aspergillus. Tests to detect aflatoxin B₁ demonstrated that this fungus is a non-aflatoxin B₁ producer, unlike A. parvisclerotigenus. Furthermore, morphological comparison with A. oryzae and A. flavus revealed that Aspergillus sp. XJ-1 belongs to A. oryzae, and named as A. oryzae XJ-1. The results of bioassays against third-instar locusts showed that mortality was dose-dependent and its median lethal concentrations were 3.3 × 10⁸, 1.7 × 10⁷ and 7.2 × 10⁶ conidia/ml on the 10th-, 13th- and 15th-day post-inoculation. Therefore, the A. oryzae XJ-1 may have biocontrol potential against locusts.     

Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The fungal entomopathogen Beauveria bassiana became established as an endophyte in coffee seedlings grown in vitro and inoculated with B. bassiana suspensions in the radicle. The fungus was recovered as an endophyte 30 and 60 days postinoculation, from stems, leaves, and roots, and at 60 days postinoculation one of the isolates was also recovered as an epiphyte. Fusarium sp., Rhodotorula sp., and four bacterial morpho-species were also detected, indicating these were present as endophytes in the seed.     

Effects of VA mycorrhiza on the growth of cacao seedlings under nursery conditions in Venezuela

The effects of vesicular-arbuscular mycorrhizae inoculation on the growth of cacao seedlings (Theobroma cacao var. Ocumare 60) grown for 5 months in a nursery were studied. The effects of introduced VAM fungi (Glomus occultum, Acaulospora appendicula, Glomus manihotis, Acaulospora morrowae and Scutellospora pellucida) in soils treated with copper oxychloride or methyl bromide, were compared with the indigenous VAM and with their respective non-inoculated controls.Cacao seedlings responded well to indigenous VAM fungi, which included Scutellospora calospora as the dominant species, inducing increases significant in height, dry weight and foliar uptake of P, Cu and Zn in relation to the sterile control. G. occultum and A. appendicula increased the height of cacao seedlings but to an extent not yet statistically significant through the duration of the experiment. S. pellucida and A. appendicula doubled the phosphorus uptake of cacao seedlings. The methyl bromide sterilization induced the lowest Cu and Zn uptake in the cacao seedlings but this effect was ameliorated with VAM inoculation. Manifest deficiency symptoms were not observed in the treatments.Copper oxychloride treatment depressed growth to the same level as the sterile control although its residual effects did not kill VAM. It could change the competitive relations among the VAM species and in this case seemed to affect adversely the more efficient native fungi.The influence of the presence and long permanence of large cotyledons in cacao on the results obtained is discussed.     

Methionine Requirements of Rats in Various Body Weights

A niaD gene encoding nitrate reductase was isolated from Aspergillus oryzae KBN616 and sequenced. The structural gene comprises 2973 bp and 868 amino acids, which showed a high degree of similarity to nitrate reductases from other filamentous fungi. The coding sequence is interrupted by six introns varying in size from 48 to 98 bp. The intron positions are all conserved among the niaD genes from A. oryzae, Aspergillus nidulans, and Aspergillus niger. A homologous transformation system was developed for an industrial shoyu koji mold, A. oryzae KBN616, based on the nitrate reductase (niaD) of the nitrate assimilation pathway.     

Production of the plant polyketide curcumin in Aspergillus oryzae: strengthening malonyl-CoA supply for yield improvement

The filamentous fungus Aspergillus oryzae was recently used as a heterologous host for fungal secondary metabolite production. Here, we aimed to produce the plant polyketide curcumin in A. oryzae. Curcumin is synthesized from feruloyl-coenzyme A (CoA) and malonyl-CoA by curcuminoid synthase (CUS). A. oryzae expressing CUS produced curcumin (64 μg/plate) on an agar medium containing feruloyl-N-acetylcysteamine (a feruloyl-CoA analog). To increase curcumin yield, we attempted to strengthen the supply of malonyl-CoA using two approaches: enhancement of the reaction catalyzed by acetyl-CoA carboxylase (ACC), which produces malonyl-CoA from acetyl-CoA, and inactivation of the acetyl-CoA-consuming sterol biosynthesis pathway. Finally, we succeeded in increasing curcumin yield sixfold by the double disruption of snfA and SCAP; SnfA is a homolog of SNF1, which inhibits ACC activity by phosphorylation in Saccharomyces cerevisiae and SCAP is positively related to sterol biosynthesis in Aspergillus terreus. This study provided useful information for heterologous polyketide production in A. oryzae.     

Using direct amplification and next-generation sequencing technology to explore foliar endophyte communities in experimentally inoculated western white pines

Fungal endophytes can influence survivability and disease severity of trees. Here we characterized the endophyte community in Pinus monticola (western white pine), an important species in the northwest USA, largely decimated by pathogenic fungi. We also assessed the ability to successfully inoculate seedlings with desirable endophytes, with the long-term goal of providing a protective microbiome and added defense from pathogens. P. monticola seedlings were inoculated in the field with potential pathogen antagonists and fungi isolated from healthy mature trees. Following inoculations direct amplification and next generation sequencing were used to characterize fungal endophyte communities, and explore interspecific competition, diversity, and co-occurrence patterns in needle tissues. Negative co-occurrence patterns between inoculated fungi and potential pathogens, as well as many other species, suggest early competitive interactions. Our study explores early endophyte community assemblage and shows that fungal inoculations may influence tree growth.     

米曲霉异源蛋白表达系统研究进展及展望

米曲霉作为一种重要的工业微生物,在异源蛋白表达方面已有广泛应用,受限于被表达蛋白的修饰及分泌过程,目前实际生产使用的基因供体主要局限于其他真菌,尤其是丝状真菌。当外源基因来源于植物、昆虫和哺乳动物时,米曲霉所生产的异源蛋白产量及生物活性往往不尽如人意。本文综述了米曲霉作为宿主表达异源蛋白的研究进展,包括其现有的遗传操作手段及异源表达方面的应用及探索,重点介绍了应用过程中面临的挑战和解决策略,另外,对米曲霉表达异源蛋白的应用前景及发展方向进行了展望。     

Diversity of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.     

Invasion of paranasal sinuses by Aspergillus oryzae

A 24-year-old male patient receiving chemotherapy for acute promyelocytic leukemia developed fever, right periorbital swelling and mild right proptosis. A head scan showed opacification of the right maxillary and ethmoid sinuses with adjacent soft tissue swelling. Biopsy of the nasal mucosa demonstrated the typical septate hyphae of Aspergillus species which was later shown on culture to be Aspergillus oryzae. A. oryzae has only rarely been reported in human disease and there is confusion as to its precise identification and role. We would like to confirm the pathogenicity of A. oryzae with this uncommon presentation of aspergillosis and also emphasize the need to take adequate and multiple cultures in suspected cases so that the possibility of species identification will be maximized.     

Improvement of Aspergillus oryzae for hyperproduction of endoglucanase: expression cloning of cmc-1 gene of Aspergillus aculeatus

FI-Carboxymethylcellulase (cmc1; family 12) is one of the endoglucanases of Aspergillus aculeatus and consists of single polypeptide chain of 221 amino acids. The cmc1 gene was expressed in Aspergillus oryzae niaD300 (niaD⁻) under promoter 8142. The plasmid pCMG14 carrying the cmc1 gene at PstI site was used as a source of the gene (920 bp) and Aspergillus oryzae was successfully transformed by the plasmid pNAN-cmc1 (harboring cmc1 gene). The plasmid was integrated in Aspergillus oryzae niaD300 genome at niaD locus and the transformed fungus constitutively produced very high amounts of endoglucanases when grown on glucose, maltose, soluble starch and wheat bran.     

Transmission of <Emphasis Type="Italic">Methylobacterium mesophilicum</Emphasis> by <Emphasis Type="Italic">Bucephalogonia xanthophis</Emphasis> for paratransgenic control strategy of Citrus variegated chlorosis

Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.