Exploring diversity rock-inhabiting fungi from northern Thailand: a new genus and three new species belonged to the family Herpotrichiellaceae.

Members of the family Herpotrichiellaceae are distributed worldwide and can be found in various habitats including on insects, plants, rocks, and in the soil. They are also known to be opportunistic human pathogens. In this study, 12 strains of rock-inhabiting fungi that belong to Herpotrichiellaceae were isolated from rock samples collected from forests located in Lamphun and Sukhothai provinces of northern Thailand during the period from 2021 to 2022. On the basis of the morphological characteristics, growth temperature, and multi-gene phylogenetic analyses of a combination of the internal transcribed spacer, the large subunit, and the small subunit of ribosomal RNA, beta tubulin and the translation elongation factor 1-a genes, the new genus, Petriomyces gen. nov., has been established to accommodate the single species, Pe. obovoidisporus sp. nov. In addition, three new species of Cladophialophora have also been introduced, namely, Cl. rupestricola, Cl. sribuabanensis, and Cl. thailandensis. Descriptions, illustrations, and a phylogenetic trees indicating the placement of these new taxa are provided. Here, we provide updates and discussions on the phylogenetic placement of other fungal genera within Herpotrichiellaceae.

First Report of Anthracnose on Giant Philodendron Caused by Colletotrichum siamense in Thailand.

Giant philodendron (Philodendron giganteum Schott) is cultivated in Thailand and has become an important ornamental houseplant with great economic value. During the rainy season in July 2022, anthracnose disease on this plant was observed at a nursery in Saraphi District, Chiang Mai Province (18°40'18" N, 99°03'17" E), Thailand. The area investigated was approximately 800 m². The disease incidence was estimated at above 15% according to the total number of plants (220 plants). The disease severity of each plant was between 25 and 50% of the necrotic lesion on the leaf. Initially, symptoms with brown spots, appeared on leaves, gradually becoming enlarged, elongate, 1 to 11 cm long by 0.3 to 3.5 cm wide, irregular, sunken, dark brown, with a yellow halo surrounding each lesion. Then, the diseased leaves eventually withered and died. Leaf pieces (5 × 5 mm2) of the margins between lesions and the healthy tissue were surface sterilized in 1% NaClO for 1 min, 70% ethanol for 30 s, and rinsed three times with sterile distilled water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in darkness. After three days of incubation, pure fungal colonies were purified by a single hyphal tip method on PDA (Korhonen and Hintikka 1980). Two fungal isolates (SDBR-CMU471 and SDBR-CMU472) with similar morphology were obtained. Fungal colonies on PDA were white and 38 to 40 mm in diameter after 3 days of incubation at 25 °C, then grayish white with cottony mycelia, the reverse side pale yellow after one week of incubation. Both isolates produced asexual structures on PDA. Setae were brown with 1 to 3 septa, 50 to 110 × 2.4 to 4.0 µm, with a cylindrical base, and acuminate tip. Conidiophores were hyaline to pale brown, septate, and branched. Conidiogenous cells were hyaline to pale brown, cylindrical to ampulliform, 9.5 to 35 µm long (n = 50). Conidia were single-celled, straight, hyaline, smooth-walled, cylindrical, ends rounded, guttulate, 9.1 to 19.6 × 3.5 to 5.6 µm (n = 50). Appressoria were brown to dark brown, oval to irregular, smooth-walled, 5 to 10 × 5 to 7.5 µm (n = 50). Morphologically, both fungal isolates resembled members of the Colletotrichum gloeosporioides species complex (Weir et al. 2012; Jayawardena et al. 2021). The internal transcribed spacer (ITS) region of the ribosomal DNA, actin (act), ß-tubulin (tub2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using primer pairs ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), T1/T22 (O'Donnell and Cigelnik 1997), CL1C/CL2C (Weir et al. 2012), and GDF1/GDR1 (Templeton et al. 1992), respectively. Sequences were deposited in GenBank (ITS: OQ699280, OQ699281; act: OQ727122, OQ727123; tub2: OQ727124, OQ727125; CAL: OQ727126, OQ727127; GAPDH: OQ727128, OQ727129). Multi-gene (combined data set of ITS, GAPDH, CAL, act, and tub2) maximum likelihood phylogenetic analyses demonstrated that both isolates were identified as C. siamense with 100% support. In a pathogenicity test, leaves of healthy plants were surface sterilized with a 0.1% NaClO solution for 3 min, rinsed three times with sterile distilled water. After being air-dried, a uniform wound (5 pores, 3 mm in width) was made at the equator of each leaf using aseptic needles. Conidial suspensions were collected from two-week-old cultures and suspended in sterile distilled water with 0.05% Tween-20. Fifteen microliters of the conidial suspension (1 × 106 conidia/ml) were placed on wounded attached leaves. As well, wounded control leaves were mock inoculated with sterile distilled water. Ten replications were conducted for each treatment and experiments were repeated twice. The inoculated plants were stored in a greenhouse at conditions of 25 to 30°C and 75 to 85% relative humidity. After 14 days, all the inoculated leaves showed disease symptoms, brown lesions with yellow halos, whereas control leaves remained asymptomatic. The pathogen C. siamense was consistently re-isolated on PDA from the inoculated tissues to complete Koch's postulates. Colletotrichum siamense has been reported as a causal agent on a wide range of host plants in Thailand and throughout the world (Farr and Rossman 2021; Jayawardena et al. 2021). Prior to this study, C. endophytica, C. karsti, C. orchidearum, C. philodendricola, and C. pseudoboninense were identified as causal agents of anthracnose on philodendrons (Xue et al. 2020; Zhang et al. 2023). However, anthracnose caused by Colletotrichum species on giant philodendron (P. giganteum) has not been previously reported. Thus, we propose C. siamense as a new causal agent of anthracnose disease on giant philodendron. This study provides information for further investigation into the epidemiology and management of this disease. Moreover, further investigations should be carried out in other philodendron growing areas of Thailand in order to specifically search for this pathogen.

Isolates of Colletotrichum truncatum with Resistance to Multiple Fungicides from Soybean in Northern Thailand.

In Thailand, four systemic fungicides-carbendazim (Car), azoxystrobin (Azo), difenoconazole (Dif), and penthiopyrad (Pen)-are commonly used to control soybean anthracnose caused by Colletotrichum truncatum; however, the pathogen has developed resistance. From 2019 to 2020, fungicide resistance in C. truncatum from fields in Chiang Rai and Chiang Mai was monitored. In tests of 85 C. truncatum isolates for resistance to multiple fungicides, 15.3% were CarRAzoR, 34.1% were triple resistant (CarRAzoRDifR or CarRAzoRPenR), and 50.6% were CarRAzoRDifRPenR. Surprisingly, all isolates tested had lost their sensitivity to one or more of the fungicides tested. The carbendazim-resistant isolates carried a point mutation in the ß-tubulin gene at codon 198 (E198A) or 200 (F200Y), and all azoxystrobin-resistant isolates had a mutation in the cytochrome b gene at codon 143 (G143A) or 129 (F129L). Moreover, a novel mutation at codon 208 (S208Y) in the gene encoding succinate dehydrogenase subunit B was detected in all of the isolates highly resistant to penthiopyrad. No mutation linked with difenoconazole resistance was detected in the genes encoding cytochrome P450 sterol 14α-demethylase. To the best of our knowledge, this is the first report of C. truncatum isolates resistant to multiple fungicides and serves as a warning to take measures to prevent the occurrence and distribution of these multiple-fungicide-resistant populations in soybean fields.

Morphology, Molecular Identification, and Pathogenicity of Two Novel Fusarium Species Associated with Postharvest Fruit Rot of Cucurbits in Northern Thailand.

Fruit rot of cucurbits caused by several pathogenic fungi has become an important postharvest disease worldwide. In 2022, fruit rot on watermelon (Citrullus lanatus) and muskmelon (Cucumis melo) was observed during the postharvest storage phase in the Chiang Mai and Phitsanulok Provinces of northern Thailand. These diseases can lead to significant economic losses. This present study was conducted to isolate the causal agent of fungi in lesions of fruit rot. A total of four fungal isolates were obtained, of which two isolates (SDBR-CMU422 and SDBR-CMU423) were obtained from rot lesions of watermelons, while the remaining isolates (SDBR-CMU424 and SDBR-CMU425) were obtained from rot lesions of muskmelons. All fungal isolates were identified using both morphological characteristics and molecular analyses. Morphologically, all isolated fungal isolates were classified into the genus Fusarium. Multi-gene phylogenetic analyses of a combination of the translation elongation factor 1-alpha (tef-1), calmodulin (cam), and RNA polymerase second largest subunit (rpb2) genes reveled that four fungal isolates belonged to the Fusarium incarnatum-equiseti species complex and were distinct from all other known species. Thus, we have described them as two new species, namely F. citrullicola (SDBR-CMU422 and SDBR-CMU423) and F. melonis (SDBR-CMU424 and SDBR-CMU425). A full description, illustrations, and a phylogenetic tree indicating the position of both new species have been provided. Moreover, pathogenicity tests were subsequently performed and the results showed that F. citrullicola and F. melonis caused symptoms of fruit rot on inoculated watermelon and muskmelon fruits, respectively. Notably, this outcome was indicative of the symptoms that appeared during the postharvest storage phase. To our knowledge, two new pathogenic fungi, F. citrullicola and F. melonis, are new causal agents of watermelon and muskmelon fruit rot, respectively. Importantly, these findings provide valuable information for the development of effective strategies for the monitoring and prevention of these diseases.

First Report of Rose Apple Leaf Spot Caused by Colletotrichum siamense in Thailand.

The rose apple (Syzygium samarangense (Blume) Merr. & L.M.Perry) plant has been commonly cultivated in Thailand. In May of 2022, leaf spot disease of rose apple was discovered in Chiang Mai Province, Thailand, with approximately 30% disease incidence. The typical symptoms initially showed brown spots (0.1 to 0.5 mm in diameter) with a yellow halo surrounding. These spots then expanded with black edges and the infected leaves appear blighted and desiccated. In humid conditions, pale yellow conidiomata formed on the lesions. Small pieces (5 × 5 mm2) of the margins between lesions and the healthy tissue were surface disinfected with 1% NaClO for 1 min, 70% ethanol for 30 s, and washed three times with sterile distilled water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25 ºC for three days. Three fungal isolates (SDBR-CMU419, SDBR-CMU420, and SDBR-CMU421) were obtained that exhibited similar morphology. Fungal colonies appeared white to gray with cottony mycelia after incubation on PDA at 25 ºC for one week. All fungal isolates produced asexual morph on PDA. Setae were 5590 × 2.53.5 µm, brown with 13-septa, cylindrical base, and tip rounded. Conidiophores were hyaline to pale brown, septate, and branched. Conidiogenous cells were hyaline to pale brown, cylindrical to ampulliform, 2050 µm long (n = 50). Conidia were one-celled, hyaline, smooth-walled, aseptate, straight, cylindrical, end round, guttulate, 1017 × 35 µm (n = 50). Appressoria were mostly formed from mycelia, oval to irregular, brown to dark brown, smooth-walled, 610 × 57 µm (n = 50). Morphologically, all fungal isolates resembled to Colletotrichum (Weir et al. 2012; Jayawardena et al. 2021). The internal transcribed spacer (ITS) region of the ribosomal DNA, actin (act), ß-tubulin (tub2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primer pairs ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), T1/T22 (O'Donnell and Cigelnik 1997), CL1C/CL2C (Weir et al. 2012), and GDF1/GDR1 (Templeton et al. 1992), respectively. The ITS (ON740892 to ON740894), act (ON759242 to ON759244), tub2 (ON759245 to ON759247), CAL (ON759248 to ON759250), and GAPDH (ON759251 to ON759253) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, GAPDH, CAL, act, and tub2) maximum phylogenetic analyses indicated that all fungal isolates clustered with C. siamense ICMP 18578 (type strain) with strong statistical (99% ML) support. For pathogenicity test, asymptomatic leaves, stems and fruits detached from healthy plants were surface disinfected using 0.1% NaClO for 3 min, washed three times with sterile distilled water, and air-dried. A uniform wound (3 pores, 1 mm in width) was made at the equator of each leaf, stem and fruit using aseptic needles. Mycelial plugs (5 mm in diameter) and conidia suspensions (1 × 106 conidia/ml) of each fungal isolate grown on PDA at 25 ºC for one week were used to inoculate both wounded and unwounded samples by the detached method (Huda­Shakirah et al. 2022; Suwannarach et al. 2022). Plugs of PDA and sterile distilled water were used as controls. Ten replications were performed for each treatment and the experiment was repeated twice. All inoculated samples were incubated in a moist chamber at 25 ºC with 90% relative humidity. The disease severity index was used to evaluate the specimens (Acar et al. 2008; Ngegba et al. 2017). After one week, both wounded and unwounded leaves that inoculated with mycelial plugs and conidia suspensions showed brown leaf spots and a weak infection. Mycelial plugs inoculated on both wounded and unwounded fruits revealed a moderate infection, but inoculation of conidia suspensions showed a weak infection. No symptoms of disease were observed on the inoculated stems. Control leaves, stems and fruits remained asymptomatic. The pathogen C. siamense was re-isolated from spot and rot lesions on PDA in order to fulfill Koch's postulates. Phoulivong et al. (2012) reported that C. siamense is a causal agent of fruit rot in rose apples cultivated in Lao and Thailand. To our knowledge, this is the first report of C. siamense causing leaf spots on rose apple plants in Thailand. Importantly, these findings will provide crucial information for epidemiologic studies and in the development of appropriate management strategies for this newly emerging disease.

Species Diversity, Distribution, and Phylogeny of Exophiala with the Addition of Four New Species from Thailand.

The genus Exophiala is an anamorphic ascomycete fungus in the family Herpotrichiellaceae of the order Chaetothyriales. Exophiala species have been classified as polymorphic black yeast-like fungi. Prior to this study, 63 species had been validated, published, and accepted into this genus. Exophiala species are known to be distributed worldwide and have been isolated in various habitats around the world. Several Exophiala species have been identified as potential agents of human and animal mycoses. However, in some studies, Exophiala species have been used in agriculture and biotechnological applications. Here, we provide a brief review of the diversity, distribution, and taxonomy of Exophiala through an overview of the recently published literature. Moreover, four new Exophiala species were isolated from rocks that were collected from natural forests located in northern Thailand. Herein, we introduce these species as E. lamphunensis, E. lapidea, E. saxicola, and E. siamensis. The identification of these species was based on a combination of morphological characteristics and molecular analyses. Multi-gene phylogenetic analyses of a combination of the internal transcribed spacer (ITS) and small subunit (nrSSU) of ribosomal DNA, along with the translation elongation factor (tef), partial ß-tubulin (tub), and actin (act) genes support that these four new species are distinct from previously known species of Exophiala. A full description, illustrations, and a phylogenetic tree showing the position of four new species are provided.