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.     

Autophagy During Conidiation and Conidial Germination in Filamentous Fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)–AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.

Addendum to:

Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

T. Kikuma, M. Ohneda, M. Arioka and K. Kitamoto

Eukaryot Cell 2006; 5:1328-36     

Synchronous Conidiation of Piricularia oryzae by the Replacement Culture

For the purpose of studying the mechanism of conidiogenesis in Piricularia oryzae, methods were developed to separate the phase of mycelial growth from that of conidiation and also to evaluate them quantitatively.

When P. oryzae was grown on media with low carbon : nitrogen ratio and high nitrogen concentration, conidiation did not take place, in spite of its vegetative growth. Conidiation occurred in a very short period of time when the above mycelia were replaced on nutritionally poor media. Cellophane membrane was overlaid on solid medium and conidia were spread uniformly. Evenly grown mycelial mat which could be easily transferred onto the post-culture medium was obtained. As the preculture medium, MSA medium with carbon: nitrogen ratio of 6.3 and nitrogen concentration of 1.5 g/liter was used. The evenly grown mycelial mat was cut into small square mats of 1.44 cm² each and the small square mycelial mats were transferred onto the post-culture medium together with the cellophane membrane. The conidiation took place in the post-culture and the vegetative growth in the preculture and the conidiation in the post-culture could be observed separately and quantitatively.

Conidiation did not occur at all in the preculture and the degree of conidiation which took place in the post-culture varied according to the precultural conditions. This means that it is a certain state of physiological condition in the preculture which determines the degree of conidiation in the post-culture. The authers designated this state of physiological condition as the “latent activity of conidiation” (LAC). For the purpose of the quantitative estimation of this activity, we expressed LAC in terms of the degree of conidiation in the post-culture under a defined cultural condition. The LAC was subject to change very easily, declined rapidly and disappeared upon prolonged preculture. Only young mycelia showed to have this activity.

The influences of the precultural condition on the development of the LAC and vegetative growth were generally parallel. However, the LAC was generally more sensitive to the environmental condition than the vegetative growth, especially to the temperature change.

The conidia formed were uniform in size and had high rate of germination. Several strains of P. oryzae tested showed very similar behavior.     

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.