Assessment of symptom induction via artificial inoculation of the obligate biotrophic fungus Phyllachora maydis (Maubl.) on corn leaves.

OBJECTIVE: Tar spot is a foliar disease of corn caused by Phyllachora maydis, which produces signs in the form of stromata that bear conidia and ascospores. Phyllachora maydis cannot be cultured in media; therefore, the inoculum source for studying tar spot comprises leaves with stromata collected from naturally infected plants. Currently, there is no effective protocol to induce infection under controlled conditions. In this study, an inoculation method was assessed under greenhouse and growth chamber conditions to test whether stromata of P. maydis could be induced on corn leaves. RESULTS: Experiments resulted in incubation periods ranging between 18 and 20 days and stromata development at the beginning of corn growth stage VT-R1 (silk). The induced stromata of P. maydis were confirmed by microscopy, PCR, or both. From thirteen experiments conducted, four (31%) resulted in the successful production of stromata. Statistical analyses indicate that if an experiment is conducted, there are equal chances of obtaining successful or unsuccessful infections. The information from this study will be valuable for developing more reliable P. maydis inoculation methods in the future.

Contour-Based Detection and Quantification of Tar Spot Stromata Using Red-Green-Blue (RGB) Imagery.

Quantifying symptoms of tar spot of corn has been conducted through visual-based estimations of the proportion of leaf area covered by the pathogenic structures generated by Phyllachora maydis (stromata). However, this traditional approach is costly in terms of time and labor, as well as prone to human subjectivity. An objective and accurate method, which is also time and labor-efficient, is of an urgent need for tar spot surveillance and high-throughput disease phenotyping. Here, we present the use of contour-based detection of fungal stromata to quantify disease intensity using Red-Green-Blue (RGB) images of tar spot-infected corn leaves. Image blocks (n = 1,130) generated by uniform partitioning the RGB images of leaves, were analyzed for their number of stromata by two independent, experienced human raters using ImageJ (visual estimates) and the experimental stromata contour detection algorithm (SCDA; digital measurements). Stromata count for each image block was then categorized into five classes and tested for the agreement of human raters and SCDA using Cohen's weighted kappa coefficient (κ). Adequate agreements of stromata counts were observed for each of the human raters to SCDA (κ = 0.83) and between the two human raters (κ = 0.95). Moreover, the SCDA was able to recognize "true stromata," but to a lesser extent than human raters (average median recall = 90.5%, precision = 89.7%, and Dice = 88.3%). Furthermore, we tracked tar spot development throughout six time points using SCDA and we obtained high agreement between area under the disease progress curve (AUDPC) shared by visual disease severity and SCDA. Our results indicate the potential utility of SCDA in quantifying stromata using RGB images, complementing the traditional human, visual-based disease severity estimations, and serve as a foundation in building an accurate, high-throughput pipeline for the scoring of tar spot symptoms.

Allocordyceps baltica gen. et sp. nov. (Hypocreales: Clavicipitaceae), an ancient fungal parasite of an ant in Baltic amber.

An ancient fungal parasite of a Camponotus ant (Formicidae: Hymenoptera) in Baltic amber is described as Allocordyceps baltica gen. et sp. nov. (Hypocreales: Clavicipitaceae). The new genus is characterized by an orange, stalked, cup-shaped ascoma with partially immersed perithecia that emerges from the rectum of the ant, two separate stromata with septate mycelium that emerge from the base of the neck and the abdomen of the ant, respectively, and free-standing putative perithecia bearing putative asci with putative multicellular ascospores fragmented into one-celled partspores. This oldest known fossil fungus of an ant could represent a precursor of the genus Ophiocordyceps, which at present is the only fungal lineage parasitizing ants of the genus Camponotus. The fossil shows unique morphological features that existed in the Hypocreales some 35-55 MYA.

Construction of Anterior Hemi-Corneal Equivalents Using Nontransfected Human Corneal Cells and Transplantation in Dog Models.

Tissue-engineered human anterior hemi-cornea (TE-aHC) is a promising equivalent for treating anterior lamellar keratopathy to surmount the severe shortage of donated corneas. This study was intended to construct a functional TE-aHC with nontransfected human corneal stromal (ntHCS) and epithelial (ntHCEP) cells using acellular porcine corneal stromata (aPCS) as a carrier scaffold, and evaluate its biological functions in a dog model. To construct a TE-aHC, ntHCS cells were injected into an aPCS scaffold and cultured for 3 days; then, ntHCEP cells were inoculated onto the Bowman's membrane of the scaffold and cultured for 5 days under air-liquid interface condition. After its morphology and histological structure were characterized, the constructed TE-aHC was transplanted into dog eyes via lamellar keratoplasty. The corneal transparency, thickness, intraocular pressure, epithelial integrity, and corneal regeneration were monitored in vivo, and the histological structure and histochemical property were examined ex vivo 360 days after surgery, respectively. The results showed that the constructed TE-aHC was highly transparent and composed of a corneal epithelium of 7-8 layer ntHCEP cells and a corneal stroma of regularly aligned collagen fibers and well-preserved glycosaminoglycans with sparsely distributed ntHCS cells, mimicking a normal anterior hemi-cornea (aHC). Moreover, both ntHCEP and ntHCS cells maintained positive expression of their marker and functional proteins. After transplantation into dog eyes, the constructed TE-aHC acted naturally in terms of morphology, structure and inherent property, and functioned well in maintaining corneal clarity, thickness, normal histological structure, and composition in dog models by reconstructing a normal aHC, which could be used as a promising aHC equivalent in corneal regenerative medicine and aHC disorder therapy.

Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders.

Successful Development of Cordyceps bassiana Stromata from Beauveria bassiana.

A specimen of Beauveria bassiana was collected from Yang-yang of Gangwon province, Korea in October 2006. Conidial isolates were prepared from the specimen by the dilution method and inoculated in brown rice medium for fruiting body production. After nearly two months incubation for perithecial stromata developed from single isolates as well as from their combinations. They were determined as Cordyceps bassiana by observing the stromatal characters and their conidial structures. This is the first report of the development of C. bassiana from B. bassiana cultures.

Cultural Characteristics and Fruiting Body Production in Cordyceps bassiana.

Single ascospore isolates of Cordyceps bassiana were observed for their colony pigmentation on Sabouraud Dextrose agar plus Yeast Extract (SDAY) plates and were inoculated in a brown rice medium for production of fruiting bodies. Colony pigmentation did not show any relationship with perithecial stromata formation. The isolates were also grown on opposite sides of SDAY agar plates and were observed for vegetative compatibility. Neither vegetative compatibility nor perithecial stromata could be found to be related to each other. It was concluded that fertile fruiting body production was independent of colony pigmentation and vegetative compatibility. Synnemata formation was found to be more common than perithecial stromata formation. This might be due to its highly conidiogenous anamorphic stage, i.e., Beauveria bassiana.

Heterothallic Type of Mating System for Cordyceps cardinalis.

Cordyceps cardinalis successfully produced its fruiting bodies from multi-ascospore isolates. However, subcultures of multi-ascospore isolates could not produce fruiting bodies after few generations. Fruiting body production also differed from sector to sector of the same isolate. Single ascospore isolates were then co-inoculated in combinations of two to observe the fruiting characteristics. Combinations of certain isolates produced perithecial stromata formation, whereas other combinations did not produce any fruiting bodies. These results show that C. cardinalis is a heterothallic fungus, requiring two isolates of opposite mating types for fruiting body production. It was also shown that single ascospore isolates are hermaphrodites.

Cultural Characteristics and Fruiting Body Production in Cordyceps bassiana

Single ascospore isolates of Cordyceps bassiana were observed for their colony pigmentation on Sabouraud Dextrose agar plus Yeast Extract (SDAY) plates and were inoculated in a brown rice medium for production of fruiting bodies. Colony pigmentation did not show any relationship with perithecial stromata formation. The isolates were also grown on opposite sides of SDAY agar plates and were observed for vegetative compatibility. Neither vegetative compatibility nor perithecial stromata could be found to be related to each other. It was concluded that fertile fruiting body production was independent of colony pigmentation and vegetative compatibility. Synnemata formation was found to be more common than perithecial stromata formation. This might be due to its highly conidiogenous anamorphic stage, i.e., Beauveria bassiana.

Successful Development of Cordyceps bassiana Stromata from Beauveria bassiana

A specimen of Beauveria bassiana was collected from Yang-yang of Gangwon province, Korea in October 2006. Conidial isolates were prepared from the specimen by the dilution method and inoculated in brown rice medium for fruiting body production. After nearly two months incubation for perithecial stromata developed from single isolates as well as from their combinations. They were determined as Cordyceps bassiana by observing the stromatal characters and their conidial structures. This is the first report of the development of C. bassiana from B. bassiana cultures.

Heterothallic Type of Mating System for Cordyceps cardinalis

Cordyceps cardinalis successfully produced its fruiting bodies from multi-ascospore isolates. However, subcultures of multi-ascospore isolates could not produce fruiting bodies after few generations. Fruiting body production also differed from sector to sector of the same isolate. Single ascospore isolates were then co-inoculated in combinations of two to observe the fruiting characteristics. Combinations of certain isolates produced perithecial stromata formation, whereas other combinations did not produce any fruiting bodies. These results show that C. cardinalis is a heterothallic fungus, requiring two isolates of opposite mating types for fruiting body production. It was also shown that single ascospore isolates are hermaphrodites.

The Progress of Biological Research of Medicinal Fungus Shiraia bambusicola / 微生物学通报

The research status is reviewed on taxonomic position, ecology, biological active matter and artificial cultivation of Shiraia bambusicola. Following problems are presented: whether there are new species and host specificity of Shiraia bambusicola. Since the artificial cultivation of stroma of Shiraia bambusicola has not been success, Shiraia bambusicola is in situation of emerge of itself and perish of itself so that it is not beneficial to resource protection and continual utilization of this medicinal fungus. It is pointed out that molecular biology of Shiraia bambusicola should be further researched and technology of artificial cultivation of Shiraia bambusicola should be explored in order to exploit and usage of Shiraia bambusicola.