Presence of Actinomycetes in agarwood tissues of Aquilaria crassna: A preliminary study

Agarwood is a valuable resin produced inside certain tree species of the family Thymalaeaceae distributed in the Asian region. Agarwood production occurs as a defense mechanism when the trees are under physical or biological stresses. However, the formation of agarwood resins in significant extractable quantities due to natural stress conditions is rare; therefore, the farmers use various methods to induce its formation artificially. Certain fungal species such as Fusarium and Aspergillus become more popular among them to produce high-quality agarwood. However, studies are rare on using other microbial organisms such as Actinomycetes, which exhibit properties of both bacteria and fungi. Among the agarwood-producing species, Aquilaria crassna is one of Asia's most commonly planted species for agarwood production. This species was introduced to Sri Lanka in 2012 for mid and lower elevations of the wet zone. Due to the lack of studies on agarwood resin formation by non-fungal microbial methods, the present study attempted to identify the presence of Actinomycetes species in agarwood resinous tissues of A. crassna. Agarwood resinous tissue samples were collected from four plantations in the wet zone of the country. Surface sterilized, small sized tissues were placed on starch casein agar medium and incubated at room temperature for ten days. Using the morphological and microscopic characteristics, it was possible to identify Nocardia, Psuedonocardia, and three Streptomyces species with varying abundance. The species level should be confirmed using molecular analysis, and their potential for agarwood resin formation inducement should be tested by re-inoculating to the healthy A. crassna trees

The genus Bipolaris

The genus Bipolaris includes important plant pathogens with worldwide distribution. Species recognition in the genus has been uncertain due to the lack of molecular data from ex-type cultures as well as overlapping morphological characteristics. In this study, we revise the genus Bipolaris based on DNA sequence data derived from living cultures of fresh isolates, available ex-type cultures from worldwide collections and observation of type and additional specimens. Combined analyses of ITS, GPDH and TEF gene sequences were used to reconstruct the molecular phylogeny of the genus Bipolaris for species with living cultures. The GPDH gene is determined to be the best single marker for species of Bipolaris. Generic boundaries between Bipolaris and Curvularia are revised and presented in an updated combined ITS and GPDH phylogenetic tree. We accept 47 species in the genus Bipolaris and clarify the taxonomy, host associations, geographic distributions and species’ synonymies. Modern descriptions and illustrations are provided for 38 species in the genus with notes provided for the other taxa when recent descriptions are available. Bipolaris cynodontis, B. oryzae, B. victoriae, B. yamadae and B. zeicola are epi- or neotypified and a lectotype is designated for B. stenospila. Excluded and doubtful species are listed with notes on taxonomy and phylogeny. Seven new combinations are introduced in the genus Curvularia to accomodate the species of Bipolaris transferred based on the phylogenetic analysis. A taxonomic key is provided for the morphological identification of species within the genus

Fungal diversity notes 1512-1610: taxonomic and phylogenetic contributions on genera and species of fungal taxa

This article is the 14th in the Fungal Diversity Notes series, wherein we report 98 taxa distributed in two phyla, seven classes, 26 orders and 50 families which are described and illustrated. Taxa in this study were collected from Australia, Brazil, Burkina Faso, Chile, China, Cyprus, Egypt, France, French Guiana, India, Indonesia, Italy, Laos, Mexico, Russia, Sri Lanka, Thailand, and Vietnam. There are 59 new taxa, 39 new hosts and new geographical distributions with one new combination. The 59 new species comprise Angustimassarina kunmingense, Asterina lopi, Asterina brigadeirensis, Bartalinia bidenticola, Bartalinia caryotae, Buellia pruinocalcarea, Coltricia insularis, Colletotrichum flexuosum, Colletotrichum thasutense, Coniochaeta caraganae, Coniothyrium yuccicola, Dematipyriforma aquatic, Dematipyriforma globispora, Dematipyriforma nilotica, Distoseptispora bambusicola, Fulvifomes jawadhuvensis, Fulvifomes malaiyanurensis, Fulvifomes thiruvannamalaiensis, Fusarium purpurea, Gerronema atrovirens, Gerronema flavum, Gerronema keralense, Gerronema kuruvense, Grammothele taiwanensis, Hongkongmyces changchunensis, Hypoxylon inaequale, Kirschsteiniothelia acutisporum, Kirschsteiniothelia crustaceum, Kirschsteiniothelia extensum, Kirschsteiniothelia septemseptatum, Kirschsteiniothelia spatiosum, Lecanora immersocalcarea, Lepiota subthailandica, Lindgomyces guizhouensis, Marthe asmius pallidoaurantiacus, Marasmius tangerinus, Neovaginatispora mangiferae, Pararamichloridium aquisubtropicum, Pestalotiopsis piraubensis, Phacidium chinaum, Phaeoisaria goiasensis, Phaeoseptum thailandicum, Pleurothecium aquisubtropicum, Pseudocercospora vernoniae, Pyrenophora verruculosa, Rhachomyces cruralis, Rhachomyces hyperommae, Rhachomyces magrinii, Rhachomyces platyprosophi, Rhizomarasmius cunninghamietorum, Skeletocutis cangshanensis, Skeletocutis subchrysella, Sporisorium anadelphiae-leptocomae, Tetraploa dashaoensis, Tomentella exiguelata, Tomentella fuscoaraneosa, Tricholomopsis lechatii, Vaginatispora flavispora and Wetmoreana blastidiocalcarea. The new combination is Torula sundara. The 39 new records on hosts and geographical distribution comprise Apiospora guiyangensis, Aplosporella artocarpi, Ascochyta medicaginicola, Astrocystis bambusicola, Athelia rolfsii, Bambusicola bambusae, Bipolaris luttrellii, Botryosphaeria dothidea, Chlorophyllum squamulosum, Colletotrichum aeschynomenes, Colletotrichum pandanicola, Coprinopsis cinerea, Corylicola italica, Curvularia alcornii, Curvularia senegalensis, Diaporthe foeniculina, Diaporthe longicolla, Diaporthe phaseolorum, Diatrypella quercina, Fusarium brachygibbosum, Helicoma aquaticum, Lepiota metulispora, Lepiota pongduadensis, Lepiota subvenenata, Melanconiella meridionalis, Monotosporella erecta, Nodulosphaeria digitalis, Palmiascoma gregariascomum, Periconia byssoides, Periconia cortaderiae, Pleopunctum ellipsoideum, Psilocybe keralensis, Scedosporium apiospermum, Scedosporium dehoogii, Scedosporium marina, Spegazzinia deightonii, Torula fici, Wiesneriomyces laurinus and Xylaria venosula. All these taxa are supported by morphological and multigene phylogenetic analyses. This article allows the researchers to publish fungal collections which are important for future studies. An updated, accurate and timely report of fungus-host and fungus-geography is important. We also provide an updated list of fungal taxa published in the previous fungal diversity notes. In this list, erroneous taxa and synonyms are marked and corrected accordingly

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper

Diversity of the Endophytic Fungal spp. in Selected Rice (Oryza sativa L) Varieties of Sri Lanka and their Hydrolytic Enzyme Producing Abilities

Rice (Oryza sativa L.) is the staple food in Sri Lanka and with increases in population, the demand for rice has also increased requiring higher yields. The use of endophytic fungal (EF) assemblages has been successful in increasing the yields in a number of crops including rice. However, the existent knowledge on the diversity among the species of EF assemblages associated with different rice varieties is hardly sufficient for this purpose. Therefore, this study was aimed at identifying and assessing the diversity of EF present in three newly improved rice varieties At 362, Bg 352, Bw 367 and one traditional variety i.e. Suwandel grown in different geographical locations/climatic zones of Sri Lanka. Healthy plant samples of each rice variety was collected during the Maha and Yala seasons in 2018/2019 from Anuradhapura, Kurunegala, Gampaha and Kalutara districts in Sri Lanka. Endophytic fungi were isolated from leaves, stems and roots of the four rice varieties using previously optimized protocols. Identifications of the isolated fungal spp. was carried out using morphological and molecular characteristics. Species were identified by PCR amplification of the Internal Transcriber Spacer (ITS) regions and comparing their sequences with those of well characterized/type strains in the National Centre for Biotechnology Information (NCBI) database. 1,920 plant segments used for isolations yielded 26 fungal genera and 39 fungal spp. The most frequently isolated and dominant species among all rice varieties were Microdochium fisheri, Dendryphiella sp and Penicillium oxalicum. Species diversity was analyzed using Shannon Wiener’s (H’) and Simpson’s dominance (1-D) indices and a high diversity of different fungal spp. were observed in Bg 352 collected from Kalutara during the Yala season. As EF have been reported to produce extra cellular enzymes as a means of showing mycoparasitic activity, selected EF isolates were screened in enriched media for production of Chitinase and Protease enzymes using standard plate assays and Glucanase enzyme production was analyzed by Dinitro Salicylic Acid assay. Out of the tested isolates, Rhizopus microsporus produced a significantly high (p≤0.05) level of chitinase, while Aspergillus fischeri showed a significantly high (p≤0.05) protease production. Penicillium oxalicum showed a significantly high (p≤0.05) glucanase production. Thus, findings of this study postulate rich EF assemblages in Sri Lankan rice varieties which could potentially be a foundation for studies on novel models of rice-fungal mutualism. Rice endophytes also produce extra-cellular hydrolytic enzymes that may contribute towards controlling rice pathogens

Associated Fungal Community in Infected Agarwood Tissues and Associated Soil of Gyrinops walla

Gyrinops walla Gaertn. is endemic to Sri Lanka and naturally populated in the forests and homegardens of lower elevations of the southwest region. It produces a highly valuable fragrant agarwood resin in stems and roots due to a self-defense mechanism, mainly to protect from fungal attacks, by activating secondary metabolic network. This study identified the fungal diversity in agarwood formed stem tissues and associated soils of G. walla trees by analysing genomic DNA of fungal isolates. Sampling of agarwood tissues and soil samples was made in G. walla growing in three different areas of the low country wet zone of Sri Lanka, vis. Neboda and Yagirala of Western Province and Rakwana of Sabaragamuwa Province. Solvent extraction was employed to extract the resin contents of agarwood samples. Surface sterilised tissues were subjected to grow, isolation and identification of fungal isolates. Soil samples were subjected to standard dilution series of plate count technique to isolate the fungi in soil samples. Morphological and molecular data were used to identify fungal isolates from infected tissues and soil samples. Phylogenetic trees were constructed to examine the relationships between the isolates sequence data and reference sequences inGenBank software. Results revealed that the agarwood resin contents of the tissue samples varied from 0.58±0.03% to 3.57±0.24%. Isolates from infected tissues of G. walla were identified as Aspergillus aculeatus, A. flavus, A. niger, A. sojae, Botryosphaeria laricina, Colletotrichum gloeosporioides, Endomelanconiopsis endophytica, Fusarium falciforme, F. proliferatum, F. solani, Nodulisporium indicum and Trichoderma harzianum. The number of fungal colony-forming units (cfug−1 ) were estimated in the soil as 2-5×108 g −1 at the reproductive stage of the G. walla trees. Among them, Ambifimbra verrucaris, A. flavus, A. niger, A. pseudonomius, A. terreus, F. falciforme, F. solani, Penicillium citrinum and P. reperi were common according to the BLAST analysis. The results confirmed that the infected tissues and soil of G. walla in the natural environment harbors multiple fungal taxa that exist in a complex system leading to agarwood production in the tree stem. Keywords: Gyrinops walla, Agarwood, Fungal community, Soil, PC

A phylogenetic and taxonomic re-evaluation of the Bipolaris - Cochliobolus - Curvularia Complex

Three genera, Cochliobolus, Bipolaris and Curvularia form a complex that contains many plant pathogens, mostly on grasses (Poaceae) with a worldwide distribution. The taxonomy of this complex is confusing as frequent nomenclatural changes and refinements have occurred. There is no clear morphological boundary between the asexual genera Bipolaris and Curvularia, and some species show intermediate morphology. We investigated this complex based on a set of ex-type cultures and collections from northern Thailand. Combined gene analysis of rDNA ITS (internal transcribed spacer), GPDH (glyceraldehyde 3-phosphate dehydrogenase), LSU (large subunit) and EF1-a (translation elongation factor 1-a) shows that this generic complex divides into two groups. Bipolaris and Cochliobolus species clustered in Group 1 along with their type species, whereas Curvularia species (including species named as Bipolaris, Cochliobolus and Curvularia) clustered in Group 2, with its generic type. The nomenclatural conflict in this complex is resolved giving priority to the more commonly used established generic names Bipolaris and Curvularia. Modern descriptions of the genera Bipolaris and Curvularia are provided and species resolved in this study are transferred to one of these genera based on their phylogeny

Fungal Planet description sheets: 154–213

Novel species of microfungi described in the present study include the following from South Africa: Camarosporium aloes, Phaeococcomyces aloes and Phoma aloes from Aloe, C. psoraleae, Diaporthe psoraleae and D. psoraleae-pinnatae from Psoralea, Colletotrichum euphorbiae from Euphorbia, Coniothyrium prosopidis and Peyronellaea prosopidis from Prosopis, Diaporthe cassines from Cassine, D. diospyricola from Diospyros, Diaporthe maytenicola from Maytenus, Harknessia proteae from Protea, Neofusicoccum ursorum and N. cryptoaustrale from Eucalyptus, Ochrocladosporium adansoniae from Adansonia, Pilidium pseudoconcavum from Greyia radlkoferi, Stagonospora pseudopaludosa from Phragmites and Toxicocladosporium ficiniae from Ficinia. Several species were also described from Thailand, namely: Chaetopsina pini and C. pinicola from Pinus spp., Myrmecridium thailandicum from reed litter, Passalora pseudotithoniae from Tithonia, Pallidocercospora ventilago from Ventilago, Pyricularia bothriochloae from Bothriochloa and Sphaerulina rhododendricola from Rhododendron. Novelties from Spain include Cladophialophora multiseptata, Knufia tsunedae and Pleuroascus rectipilus from soil and Cyphellophora catalaunica from river sediments. Species from the USA include Bipolaris drechsleri from Microstegium, Calonectria blephiliae from Blephilia, Kellermania macrospora (epitype) and K. pseudoyuccigena from Yucca. Three new species are described from Mexico, namely Neophaeosphaeria agaves and K. agaves from Agave and Phytophthora ipomoeae from Ipomoea. Other African species include Calonectria mossambicensis from Eucalyptus (Mozambique), Harzia cameroonensis from an unknown creeper (Cameroon), Mastigosporella anisophylleae from Anisophyllea (Zambia) and Teratosphaeria terminaliae from Terminalia (Zimbabwe). Species from Europe include Auxarthron longisporum from forest soil (Portugal), Discosia pseudoartocreas from Tilia (Austria), Paraconiothyrium polonense and P. lycopodinum from Lycopodium (Poland) and Stachybotrys oleronensis from Iris (France). Two species of Chrysosporium are described from Antarctica, namely C. magnasporum and C. oceanitesii. Finally, Licea xanthospora is described from Australia, Hypochnicium huinayensis from Chile and Custingophora blanchettei from Uruguay. Novel genera of Ascomycetes include Neomycosphaerella from Pseudopentameris macrantha (South Africa), and Paramycosphaerella from Brachystegia sp. (Zimbabwe). Novel hyphomycete genera include Pseudocatenomycopsis from Rothmannia (Zambia), Neopseudocercospora from Terminalia (Zambia) and Neodeightoniella from Phragmites (South Africa), while Dimorphiopsis from Brachystegia (Zambia) represents a novel coelomycetous genus. Furthermore, Alanphillipsia is introduced as a new genus in the Botryosphaeriaceae with four species, A. aloes, A. aloeigena and A. aloetica from Aloe spp. and A. euphorbiae from Euphorbia sp. (South Africa). A new combination is also proposed for Brachysporium torulosum (Deightoniella black tip of banana) as Corynespora torulosa. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa