Potato Dextrose Agar Research Articles

Soil amendments with nanoparticles control cucumber wilt caused by Fusarium oxysporium

Management of Fusarium wilt, a destructive plant disease, mainly relies on chemicals, but research on novel and eco-friendly methods is needed due to the hazardous effects of chemical pesticides. This study explored the soil application of zinc oxide nanoparticles (ZnONPs) to manage Fusarium wilt disease in cucumber plants. The antifungal activity of ZnONPs against in-vitro growth of Fusarium oxysporum was tested through food poisoning test on the potato dextrose agar medium. The in-vivo potential of NPs for managing Fusarium wilt was checked in pot experiment. In-vitro results confirmed the concentration-dependent in-vitro antifungal activity. The NPs application at 500 µg/ml and 600 µg/ml concentrations showed phytotoxicity and significantly inhibited seed germination and growth of cucumber plants. However, NPs at 300 µg/ml significantly suppressed the pathogenic effects of F. oxysporum on cucumber plants without any phytotoxic effect. The pathogen-inoculated cucumber plants grown in soil amended with NPs showed significantly enhanced growth, lower disease severity, and improved root physiology compared to untreated plants. The plants under NPs treatment showed higher chlorophyll and nitrogen content while decreased membrane permeability and malondialdehyde content. Along with direct antifungal activity, the application of NPs caused an increased expression of defense-related genes and higher activities of defense-related enzymes. These findings verified the efficiency of ZnONPs for suppressing Fusarium wilt disease in cucumber crops, thereby promoting a non-chemical and environmentally friendly approach to controlling Fusarium wilt disease.

First Report of Alternaria alternata Causing Leaf Spot on Catalpa bungei in China.

Catalpa bungei originates from China. It is fast-growing and possesses a vertically aligned trunk, rendering it a commendable construction material and a significant economic species. In July 2022, a serious leaf spot occurred in the LanLake farm (surveyed area of about 700 acres) in Nanyang (33°3'23" N, 112°28'50" E), Henan Province, China. The incidence rate of leaf disease reached 54% (n=100). The disease initially manifested as irregular round spots with a yellowish-brown hue, subsequently extending in all directions. Later, the lesion periphery exhibited a darkening effect, leading to yellowing. Twenty diseased leaves were randomly collected and cut into small pieces at the interfaces between infected and healthy tissues. The tissues were sterilized in a solution of 75% ethanol and 1% NaClO for 30 seconds and 1 minute, respectively. After rinsing in sterile water, the pieces were placed on potato dextrose agar (PDA) plates and incubated at 25°C for 5 days. A total of 29 purified fungal strains were acquired, exhibiting comparable phenotypes in terms of morphological characteristics. Three strains (QS1-1, QS1-2, and QS1-3) were isolated for subsequent investigations. The colony exhibited abundant aerial mycelium with shades ranging from dark green to grey-brown on the reverse side. To analyze the morphological characteristics of conidia, potato carrot agar (PCA) was used as the culture medium and incubated at 25°C with a 12-hour light/dark cycle. Conidia were obclavate or spheroidal, dark brown, with 3 to 5 transverse septa, and 1 to 4 longitudinal septa, measuring 12.4 to 36.7 × 4.4 to 9.0 μm (n=100), with conical beak lengths ranging from 0 to 4.3 μm. These morphological traits suggested that the pathogen shares similarities with the Alternaria species. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (tef1), glyceraldehyde 3-phosphate dehydrogenase gene (gapdh), and RNA polymerase II second largest subunit (rpb2) were amplified for further molecular identification. The resultant sequences were submitted to GenBank with the following accession numbers: OR733559, OR742124, OR761873 (ITS), OR939796, OR939797, OR939798 (tef1), OR939801, OR939802, OR939803 (gapdh), and PP054846, PP054847, PP054848 (rpb2). A Phylogenetic tree was constructed of combined genes (ITS, tef1, gapdh, and rpb2) of sequences, alongside the sequences of the type strains by the neighbor-joining method. The three strains formed a clade with the strains CBS 121456 of Alternaria alternata in phylogenetic trees, being separated from other Alternaria spp. The morphological features and molecular analyses supported the strains as members of Alternaria alternata (Woudenberg et al. 2015). To validate pathogenicity, a conidial suspension (106 conidia ml-1) of all three strains was inoculated onto three healthy leaves of five seedlings, with 50 μl of inoculum absorbed with cotton balls. Another group of five plants received sterile water as a control. All plants were incubated in a climate chamber at 28°C and 90% relative humidity. Four days post-inoculation, lesions resembling natural phenomena were observed, whereas control plants showed no symptoms. Subsequent reisolation produced cultures that were morphologically and molecularly identical to the original strains, fulfilling Koch's postulates. Stem canker of C. bungei caused by Phytophthora nicotianae has been reported in China (Chang et al. 2022). This is the first report of A. alternata causing leaf spots on C. bungei in China. Further research is required on management options to control this disease and the host range still needs to be clarified for accurate disease management.

A Study on the Valorization of Rice Straw into Different Value-Added Products and Biofuels

This work depicts that rice straw (RS), which is one of the major lignocellulosic wastes all over the world and causing many environmental problems, has considerable amounts of protein, ash, macronutrients, and micronutrients of approximately 11.38%, 16.77%, 2.27 mg/kg, and 771.9 mg/kg, respectively; besides, a C/N ratio of 15.18, a total N, P2O5, and K2O content of 1.85%, and a considerably low concentration of undesirable heavy metals and silica of approximately 77.69 mg/kg and 109 mg/kg are also present, which recommends its applicability as a precursor feedstock for the production of organic fertilizer and animal fodder. The batch solid-state fermentation (SSF) of RS by Trichoderma longibrachiatum DSMZ 16517 produced considerable amount of total reducing sugars (TRS) of approximately 339.2 mg TRS/g RS under the optimum operatic conditions of 20% (w:v) substrate concentration, pH 7, 1% inoculum size, a 9-day incubation period, and 30°C incubation temperature. The readily available and cost-effective agroindustrial waste, sugarcane molasses, proved to enhance the fungal biomass growth and (hemi) cellulolytic enzymes activities. The inoculated RS-SSF batch process with T. longibrachiatum precultured on 10% molasses enhanced the (hemi) cellulolytic enzymatic activities and TRS production rate by approximately 5.82 and 3.8 folds, respectively, relative to that inoculated by T. longibrachiatum precultured in the conventional potato dextrose broth medium. The separate hydrolysis and fermentation processes by different yeast strains Candida tropicalis DSM 70156, C. shehatae ATCC 58779, and Saccharomyces cerevisiae ATCC 64712 revealed an efficient bioethanol yield and productivity that ranged between 0.36 and 0.38 g/g sugars and 0.22 and 0.23 g/L/h, respectively, with concomitant competent fermentation efficiencies that ranged between 48.35% and 51.25%. The proximate analysis of rice straw before and after fungal hydrolysis proved calorific values of approximately 15.8 MJ/kg and 16.05 MJ/kg, respectively, recommending their applicability as primary and secondary solid biofuels. Thus, this study proved the waste prosperity of RS for environmental opulence and sustainability.

First repot of Chaetomium globosum causing leaf spot on Averrhoa carambola in India.

The Star fruit (Averrhoa carambola L.) was globally distributed, particularly in countries like China, India, Indonesia and was renowned for its abundant vitamin, mineral and antioxidant content (Reddy et al., 2023). In early February 2023, leaf spot symptoms were observed on A. carambola at 2 hectare model orchard, College farm, Agricultural College, Aswaraopet (17.252038 latitude, 81.109574 longitude) and Horticulture nurseries of Aswaraopet, Bhadradri Kothagudem (Dist), Telangana, India. In the surveyed fields (February-2023 to February 2024), the disease was prevalent year round, with varying incidence i.e., July to February (35% to 40% with a yield loss of 35%) and from March to June (20% to 30% with a yield loss of 20%). The disease was initiated as small reddish spots, which grew over 8-10 days to 1-5 mm spots with a necrotic center, dark reddish brown margin and a prominent yellow halo around them, within 17 to 20 days, all spots coalesced, resulting in leaf yellowing and defoliation (SF 1). To isolate pathogen, diseased leaf tissues (n=20) (5 × 5 mm) were surface sterilized (70% alcohol (30 s), 1% sodium hypochlorite (30 s) and sterile distilled water (3 × 60 s), inoculated to PDA media and incubated at 26 ± 2°C with 12 hours photoperiod for 72 hours (Chi et al. 2022). The emerging hyphae from the diseased tissues were sub cultured and incubated on PDA at 26 ± 2 °C. Initially, the fungal colonies appeared white, later transitioning to light brown and finally developed into olivaceous grey colour (SF 2A). The ascospores (n=20) were lemon shaped, pointed at both ends, with a length of 10.3 μm (9.1 to 12.1 μm) and width of 8.6 μm (7.2 to 10.2 μm) (SF 2B and 2C). For further identification of the pathogen, four fungal isolates were cultured in potato dextrose broth and genomic DNA was extracted using the CTAB method. Identification of the pathogen was confirmed through amplification and sequencing the Internal Transcribed Spacer region (ITS), Translation Elongation Factor 1-α (TEF1) and RNA polymerase subunit (RPB1) genes. The resulting sequences were deposited in Gen Bank with accession numbers (OR337915, OR337916, OR337893 and OR337892 for ITS, OR669280, OR669281, OR669282, and OR669283 for TEF, and PP092153, PP092154, PP092155 and PP092156 for RPB1). To study pathogenicity of fungus, five isolates of C. globosum were isolated from five A. carambola plants, grown in potato dextrose broth. Spore suspension of 1x106 spores/mL were prepared by adjusting with hemacytometer and were sprayed onto the leaves of healthy, surface sterilized (50% ethanol), 3 months old A. carambola plants and incubated in greenhouse (T: 26°C; RH: 80%). For each of the five isolates, the spore suspension from each individual isolate was inoculated into three plants and three control plants were maintained for each isolate. The experiment was replicated thrice. After a period of 10 to 12 days, symptoms appeared on the inoculated leaves in the form of reddish spots, similar to original symptoms (Alam et al. 2021) (SF 1H). The fungus isolated from the inoculated leaves was morphologically similarities to C. globosum. Notably, C. globosum, a widespread leaf spot pathogen in crops like A. hypogaea, C. sativa and Pomegranate (Chaffin et al. 2020). To our knowledge, this is the first report of A. carambola leaf spot caused by C. globosum in India and worldwide. The result will be helpful for providing a basis for further research on the control of the disease.

First Report of Alternaria alternata Causing Postharvest Fruit Rot of Indian Jujube (Ziziphus mauritiana) in China.

Ziziphus mauritiana Lam., commonly known as Indian jujube or ber, is a popular fruit crops grown in tropical and sub-tropical regions of China. It is commonly stored at 4℃, relative humidity of about 90%, combined with waxing or sealing with film bag. In January 2023, a postharvest fruit rot was observed on Indian jujube in three markets located in Nanchang city of Jiangxi province, China, with a disease incidence of 4 to 10%. Initially, brown spots appeared on the surface or base of thefruit, which gradually expanded into irregular brown lesions. Gray-white hyphae developed in the center of the lesions, and ultimately thefruitrotted. To isolate the pathogen, small pieces (5 × 5 mm) of ten infected fruits were surface-sterilized in 75% ethanol for 15 s and then 1% sodium hypochlorite for 30 s, rinsed three times in sterile water, plated onto potato dextrose agar (PDA), and incubated at 25°C for 3 days. Eight strains with similar morphological characteristics were isolated, and one representative isolate (JXAA-1) was used for morphological and molecular characterization. The colonies on PDA were initially olive green with white margins, and later turned dark olive or black with profuse sporulation. Conidia were borne singly or in a chain, brown, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, obclavate to obpyriform, and measured 12.9 to 33.7 × 7.5 to 12.9 μm (n = 30). On the basis of morphological characteristics, the isolates were tentatively identified as Alternaria spp. (Simmons 2007). To confirm the identification, genomic DNA was extracted from the isolate JXAA-1 with the Fungi Genomic DNA Extraction Kit (Solarbio Biotech, China). The 18S nrDNA (SSU), 28S nrDNA (LSU), internal transcribed spacer of the rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (TEF1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene regions (OPA 10-2) were amplified and sequenced using primers NS1/NS4, LR7/LR0R, ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, Alt-for/Alt-rev, PG3/PG2b, OPA10-2L/OPA10-2R, respectively (Woudenberg et al. 2015). The obtained DNA sequences (SSU: PP190241; LSU: PP190242; ITS: PP189927; GAPDH: PP196557; TEF1: PP196558; Alt a 1: PP196559; EndoPG: PP196560; and OPA 10-2: PP196561) showed 100% homology with those of A. alternata (GenBank accession nos. MT000349 [1020/1020 bp]; KP940477 [1312/1312 bp]; MK972909 [583/583 bp]; MN615421 [593/593 bp]; MN046379 [280/280 bp]; MN304714 [490/490 bp]; MN698284 [458/458 bp] and MH975214 [701/701 bp]). A maximum likelihood phylogenetic tree was constructed by combining all sequenced loci in IQTREE web servers. The isolate JXAA-1 clustered with Alternaria alternata (CBS 121336). The fungus associated with postharvest fruit rot on Z. mauritiana was thus identified as A. alternata. To evaluate the pathogenicity, six surface sterilized fruits were wounded by a sterile scalpel and inoculated with a 10 μl drop of spore suspension (1 × 105 conidia/ml) of isolate JXAA-1. Another six fruits were inoculated with sterilized ddH2O as control and the experiment was repeated three times. All fruits were incubated at 25°C and 80% relative humidity. After 5 days, all the wounded fruit inoculated with A. alternata showed similar symptoms to those observed previously, while the control fruits remained healthy. A. alternata was consistently reisolated from infected fruit and confirmed by morphological and molecular data, fulfilling Koch's postulates. A.alternata has previously been reported causing leaf spot and fruit rot on Chinese jujube (Ziziphus jujuba) in China (Bai et al. 2015; Li et al. 2021). But to our knowledge, this is the first report of A. alternata causing postharvest fruit rot on Indian jujube (Z. mauritiana) in China. Therefore, managers should pay more attention to postharvest fruit rot of jujube caused by A. alternata, the foam bag is put on after the membrane bag is sealed, the broken or infected fruit is picked out in time to reduce the spread of pathogenic fungus.

First report of Neocosmospora pisi causing blight on Ammopiptanthus mongolicus in China.

Ammopiptanthus mongolicus is the only evergreen broad-leaved shrub in the desert region of Northwest China, which is one of the dominant species in the desert vegetation of the region, playing an important role in maintaining the stability of the local desert ecosystem. A. mongolicus is also very hardy and drought resistant and can survive extreme temperatures (Liu et al. 2013; Yang et al. 2022). The large-scale death of A. mongolicus could cause desertification in the region. Two months after the discovery of Fusarium verticillioides causing blight on A. mongolicus in Etuoke county, Inner Mongolia Autonomous Region in September 2023 (Yang et al. 2024), a large number of A. mongolicus plants with symptoms of blights were found in Lingwu city, Ningxia Hui Autonomous Region, China (106.442368°E, 37.734026°N) in November 2023. The incidence of diseased plants in this field was about 30%. The field symptoms in Lingwu city were similar to those observed in Etuoke county. The diseased leaves initially turned yellow, then wilted and dehisced, eventually resulting in plant death (Figure 1). The roots of the diseased plants were cut diagonally and the central cylinder showed a brown color (Figure 2). In order to investigate whether the death of A. mongolicus was caused by the same pathogen as those identified previously, 30 roots were collected from 10 diseased plants. After rinsing and surface sterilization (70% ethanol for 3 min and 2.5% NaClO for 5 min, rinsed 3 times with sterile distilled water), diseased tissues (10×10 mm) were placed on potato dextrose agar (PDA) (3 pieces per plate) and incubated from 3 to 5 days at 25°C. The strain AmP5 was isolated and used for further study. After 3 days on PDA medium, fungal colonies were white to milky, the undersides of the cultures were yellowish to orange-brown (Figure 3). After 7 days on synthetic nutrient-poor agar (SNA), microconidia were ovoidal or with a rounded apex and truncate base, 10.5 ± 1.5 μm × 1.6 ± 0.2 μm (×400). The macroconidia were slightly curved or arcuate, 40.5 ± 3.5 μm × 5 ± 0.5 μm (×400) (Figure 4) (Šišić et al. 2018). The pathogen was confirmed to be Neocosmospora pisi by multigene phylogenetic analysis of TEF, RPB1 and RPB2 genes using primers EF1/EF2, F5/G2R and 5F2/11AR, respectively (O'Donnell et al. 2022). The sequences of PCR products were deposited in GenBank with accession numbers OR944631 (RPB1), OR988086 (TEF) and OR988087 (RPB2), respectively. The results of pairwise alignment in Fusarioid-ID database (Crous et al. 2021) showed 99.84% similarity and 83.96% overlap of the EF1-α sequence to the corresponding sequence LR583636 of ex-epitype CBS 123669 of Neocosmospora pisi (syn. Fusarium solani f. sp. pisi), 99.72% similarity and 85.66% overlap of the RPB1 sequence to the corresponding sequence MW834242 of ex-epitype CBS 123669 of N. pisi, and 99.47% similarity and 78.26% overlap of RPB2 sequence to the corresponding sequence LR583862 of ex-epitype CBS 123669 of N. pisi. Moreover, the result of polyphasic identification in the Fusarioid-ID database also showed EF1-a, RPB1, and RPB2 sequences had 99.15% similarity to the corresponding sequences of CBS 1233669. The pathogenicity of AmP5 was tested on potted 64 days old seedlings A. mongolicus plants. The roots of 3 seedlings were inoculated with conidial suspension (1×106 /ml), and another 3 used as controls were inoculated with sterile water, by gently peeling off the soil around the roots during inoculation, and pouring the conidial suspension around the roots (10 ml/seedling). All plants were placed in a growth chamber at 18-25℃ (10 h light; 14 h dark). After incubation for 3-5 days, the symptoms similar to those observed in the field (Figure 5), including brown rot of steles (Figure 6), developed on plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The same pathogen was reisolated from inoculated roots and confirmed as N. pisi based on morphological and molecular analyses (TEF, RPB1 and RPB2). To our knowledge, this is the first report of blight on A. mongolicus caused by N. pisi in China. This study also indicates that blight on A. mongolicus can be caused by different fungal pathogens. Blight caused by different pathogens may have different in terms of control measures and pathogenic mechanisms, so the study of blight caused by different pathogens is of profound value.

First Report of Paramyrothecium breviseta Causing Target Spot on Amorphophallus muelleri in Yunnan, China.

Amorphophallus muelleri is an Araceae plant with perennial tuber, widely used in food, pharmaceutical and chemical industry due to its richness in glucomannan. In April 2022, an outbreak of a target spot on A. muelleri plantlets was observed in a nursery in Ruili, Yunnan, China. The leafstalks of the diseased plantlets in the nursery turned brown and decayed (Fig.1 A-B), then gradually some water-soaked spots on the true leaves developed along the veins (Fig.1 A). Subquencely, the spots on the true leaves turned dark green to white-grayish in the center, which formed light to dark brown concentric rings with a target-like appearance surrounded by a yellow halo (Fig.1 C). When the temperature was 20-34℃ and the relatively humidity was 25-80%, dark-green to black sporodochia with white hypha appeared on the lower and upper leaf surfaces. Finally, 5-8% of the plants surveyed on 800 m2 of one-year-old plantlets in the nursery showed the symptoms and some plants with infected leafstalks would be death. Similar symptoms were also observed on about 10% of the transplanted plants surveyed on 12000 m2 (1.2 ha) of two-year-old plantlets in the field. Five diseased leaves from five distinct plantlets in the nursery were collected for pathogen isolation. Leaf pieces(5 x 5 mm) were cut from the edge of necrotic lesions, and surface-sterilized with 2.5% sodium hypochlorite for 1 min, 75% ethanol for 30 s, then rinsed 5 times by sterilized distilled water, finally put the leaf pieces on sterilized filter paper for 3-5 minutes to dry them and transferred onto potato dextrose agar (PDA) in petri dishes at 25℃ for three days. Five pure cultures identical to colony and conidial characteristics were isolated from five individual plants. The representative pure culture (M1) was grayish-white and circular colonies were 7.50 cm in diamter after 15 days at 25℃, with dark green concentric rings of sporodochia, the dorsal view of the colonies were yellowish. Conidia were aseptate, smooth, cylindrical, 5.00-6.25 (5.71) x 1.25-1.67 (1.63) µm (n = 20) rounded at both ends. A spore suspension (1 x 106 spores/ml) was prepared by harvesting spores from 15-day-old cultures grown in the dark at 25℃, then a thirty-ml of spore suspension was sprayed on the healthy leaves of 10 two-year-old plantlets. Thirty-ml of sterile water was sprayed on the healthy leaves of another 10 seedlings and used as the control. All seedlings were placed in a nursery at 20 to 34℃ and a relative humidity of 25 to 80%. Similar symptoms (Fig.1 D-F) to those observed in the nursery and field developed on all the 10 seedlings inoculated with M1 after two days, but not on the control leaves. The pathogenicity tests were repeated for three times. Fungal cultures reisolated from the infected leaves were identical to the original colonies and conidia, completing Koch's postulates. The internal transcribed spacer (ITS, primers ITS1 and ITS4) region of ribosomal DNA (OQ553785), calmodulin (cmdA, primers CAL-228F and CAL2Rd)(OQ559103), RNA polymerase II second largest subunit (rpb2, primers RPB2-5F2 and RPB2-7cR) (OQ559104) and β-tubulin (tub2, primers Bt2a and Bt2b) (OQ559105) of M1 had 100%, 98.52%, 98.98% and 98.98% identity with the sequences of Paramyrothecium breviseta CBS544.75 (KU846289 for ITS, KU846262 for cmdA, KU846351 for rpb2, and KU846406 for tub2), respectively. In the phylogenic tree based on ITS, cmdA, rpb2 and tub2 gene sequences, the pure culture M1 clustered with P. breviseta CBS544.75, SDBR-CMU387, DRL4 and DRL3, which has been reported as the pathogen of leaf spot of Coffea arabica in China, C. canephora in China and Thailand (Wu et al. 2021; Withee et al. 2022). Molecular and morphological observations showed the pure culture M1 were P. breviseta (Withee et al. 2022), in addition the disease was named as target spot dueing to the typical target symptom on the leaves. To our knowledge, this is the first report of P. breviseta on A. muelleri from Yunnan, China, as well as worldwide. This disease can caused serious economic losses of A. muelleri dueing to that it can result 5-8% death of the plants in the nursery.

First Report of Anthracnose Leaf Spot Caused by Colletotrichum americae-borealis on Potentilla anserina in China.

Potentilla anserina L. has an abundance of bioactive compounds and is widely recognized for its diverse applications in traditional medicine and as a food. In August 2023, typical symptoms of anthracnose were observed in 80% of P. anserina plants in Harbin, China. Symptoms, characterized by reddish-brown spots, tend to occur more frequently on leaves closer to the ground. They initially appeared as oval or irregular circles, measuring 1 to 3 mm in diameter, and later merged into larger patches surrounded by chlorotic areas on the leaves. Twenty leaves exhibiting characteristic symptoms were sampled. Each leaf was sectioned into 5×5 mm pieces at the interface between the diseased and healthy tissues. The sections were disinfected sequentially with 75% ethanol for 30 s, followed by 1% NaClO for 2 min, rinsed three times in sterilized distilled water. Post air-drying, samples were cultured on potato dextrose agar (PDA) plates and incubated at 26°C in the dark for 5 d, yielding nine morphologically similar single-spore isolates (JTC1 to JTC9). The colonies initially displayed gray aerial mycelia, becoming pale brown, accompanied by numerous black microsclerotia. The acervuli appeared black, protruded from the surface of the medium, and were adorned with dark brown setae. Setae (n=50) ranged from 58.4 to 188.2 μm in length, appearing dark brown to black, with smooth walls, rounded tips, swollen bases, and containing 1 to 4 septa. The conidia were hyaline, aseptate, cylindrical to spindle-shaped, with blunt and rounded ends, measuring 13.7 to 18.3 μm in length and 3.4 to 4.3 μm in width (n=50). Morphological analysis indicated a close affinity with Colletotrichum americae-borealis (Damm et al. 2014). For molecular identification, genomic DNA was extracted from three representative isolates (JTC1, JTC2, and JTC3).The ITS, HIS3，GAPDH, and ACT genes were amplified and sequenced using the primers described previously by Damm et al. (2014). The sequences were submitted to GenBank (ITS: PP338190 to PP338192; HIS3: PP355770 to PP355772; GAPDH: PP355773 to PP355775; ACT: PP355776 to PP355778). BLAST analysis showed 99 to 100% identity with C. americae-borealis type strain CBS 136232 (GenBank accessions: KM105224, KM105364, KM105579, and, KM105434, respectively). Multigene phylogenetic analysis positioned the three isolates close to C. americae-borealis. Pathogenicity tests were performed twice on 6-week-old P. anserina seedlings (cv. Qinghai Juema 1) in a greenhouse. A conidial suspension of the JTC1 isolate (1×10⁵ conidia/ml) was sprayed applied to ten pots, each containing two seedlings, and the plants in the control pots were sprayed with sterile distilled water. Two weeks after inoculation under greenhouse conditions (26/22°C day/night temperature, 12-hour photoperiod, 90% relative humidity), the inoculated seedlings exhibited brown spots and necrotic lesions similar to those observed in the field, C. americae-borealis was successfully reisolated from these symptomatic tissues. To the best of our knowledge, this is the first report of C. americae-borealis causing leaf spot on P. anserina in China. Anthracnose caused by C. americae-borealis is associated with leaf spot disease in oats (Wang et al. 2022), alfalfa (Li et al. 2021), and licorice (Lyu et al.2020). However, C. americae-borealis poses a significant threat to P. anserina in China as well, highlighting the urgent need to develop effective disease management strategies.

First Report of Calosphaeria pulchella causing Canker and Twig Dieback of Peach (Prunus persica) in California, U.S.A.

California leads the United States in peach (Prunus persica L.) production, with approximately 505,000 tons produced in 2021 and valued at $378.3 million (California Agriculture Statistics Review, 2021-2022). During the spring and summer of 2023, twig and branch dieback were observed in three peach orchards (cvs. Late Ross and Starn) in San Joaquin County, California. Wood cankers and discoloration also occurred in branches, generally initiating at pruning wounds. Approximately 8 symptomatic twigs or branches per orchard were collected to proceed with the isolation of necrotic tissues on acidified potato dextrose agar (APDA). Isolations consistently yielded colonies of the fungal pathogen Calosphaeria pulchella (Pers. : Fr.) J. Schröt. (Réblová et al. 2004; Trouillas et al. 2012). Pure cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. Colonies on APDA grew dark pink to red or purple in their center, with a white margin. Conidiogenesis was phialidic, producing round conidial masses at the tip of phialides. Conidia were produced abundantly on APDA, and were hyaline, allantoid to oblong-ellipsoidal, 4 to 5.5 (7) × 1.2 to 2.3 μm (n = 60). Two representative isolates (SJC-62 and SJC-64) were selected for genomic DNA extraction and sequencing of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers and the beta-tubulin (TUB2) gene region using primers Bt2a and Bt2b. Consensus sequences of the two genes for the two isolates (ITS: PP063990, PP063991; TUB2: PP068303, PP068304) were compared to reference sequences (Réblová et al. 2015; Trouillas et al. 2012) using BLAST analysis. The ITS sequences of SJC-62 and SJC-64 were 99.8 and 99.5% identical to that of C. pulchella ex-type strain CBS 115999 (NR145357) and reference strain SS07 (HM237297); the TUB2 sequences were at least 98.5% identical to that of C. pulchella CBS 115999 (KT716476). Pathogenicity tests were conducted in 2- to 3-year-old healthy branches on 7-year-old peach trees, cvs. Loadel, Late Ross and Starn using the two fungal isolates and a control treatment (1 branch per treatment and 3 branches per tree) on each of 8-tree replicates. Branches were inoculated in June 2023 following wounding with a 5 mm cork borer to remove the bark and placing an agar plug from the margin of 10-day-old colonies on APDA directly into the fresh wound. Sterile agar plugs were used as controls. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to retain moisture. The experiment was completed twice. After four months, cankers and vascular discolorations developed around the inoculation sites. Length of vascular discoloration in inoculated branches averaged 72, 75, and 79 mm, for the Loadel, Starn, and Late Ross cvs., respectively. Calosphaeria pulchella was re-isolated from inoculated branches at 80 to 100% recovery rate, thus fulfilling Koch's postulates. The average length of vascular discoloration in the control was 13.5 mm and no fungi were recovered from control branches. Calosphaeria canker caused by C. pulchella is a global disease of sweet cherry. Recently, it was reported to cause cankers in peach trees in Chile (Grinbergs et al. 2023). To our knowledge, this is the first report of C. pulchella causing cankers and twig dieback of peach trees in the United States. These findings improve our knowledge of the etiology of canker diseases affecting peach trees and is critical for the development of effective disease management strategies.

Mycological Assessment of Selected Swimming Pools of Recreational Centers around the University of Port Harcourt, Nigeria

Aims: This study was carried out to determine the physicochemical properties and mycological safety of four swimming pools within and around the University of Port Harcourt, Rivers State, Nigeria. Study Design:  Random sampling design was employed. Place and Duration of Study: Department of Microbiology Laboratory, University of Port Harcourt, between August and October, 2023. Methodology: A total of 8 samples were collected before and after patronage by patrons from 4 swimming pool. Standard methods were employed for the physicochemical parameters such as pH, turbidity and residual chlorine as well as the enumeration for fungi using potato dextrose agar. Results: The pH values ranged from 4.50 to 10.00. The Nephelometric Turbidity Unit ranged from 0.45 to 0.90 NTU while the residual chlorine ranged from 0.36 to 355.00 mg/l. The fungal counts ranged from 1 to 9 cfu/ml, while the isolated species on the basis of cultural morphology and microscopic characteristics were identified as species of Fusarium, Penicillium, Aspergillus, Histoplasma, Rhodotorula, Microsporium and Trichophyton. Conclusion: This study has revealed a potential health risk associated with the use of water of the examined recreational facilities due to none compliance with the WHO standard of < 1 cfu/100ml for fungal and the other parameters, suggesting a public health concern and need for effective interventions.

First Report of Neofusicoccum parvum Causing Fruit Rot on Psidium guajava in China.

Guava (Psidium guajava L.), a nutrient-rich and economically significant fruit, is extensively cultivated in southern China. In six continuous years (from 2018 to 2023), dark-purple rotted guava with sunken lesion were observed on guava trees (variety 'Zhenzhu', aged over 5 years) in Dongguan and Panyu districts, Guangdong Province. Annually, the incidence of fruit rot disease in the affected fields reached 30% to 50% and significantly reduced the yield and quality of harvest guava. The initial symptoms on the epicarp of the fruits were black, needle-like dots that rapidly spread, causing partial or complete fruit rot within two to three weeks. To identify the causative agent, six symptomatic fruits were collected from two different orchards. Samples of 0.5 cm³ were excised from the lesion margins of each fruit. These samples were surface-sterilized with 70% ethanol for 30 seconds, followed by 0.2% NaClO for 2-3 minutes, and rinsed in sterile water three times. The samples were then cultured on potato dextrose agar (PDA) at 25°C for five days. This process yielded eight fungal isolates with similar morphological. Initially, the colonies were white with dense aerial mycelium becoming dark gray after 4 to 5 days. The mycelia were septate and branched. No spores were observed on PDA. To induce spore formation, the isolates were cultured on water agar for 20 days. This process led to the production of hyaline, aseptate, ellipsoid conidia, which were thin-walled, smooth-surfaced, and measured 3.7-5.1 × 1.6-2.2 μm (n = 100). Three isolates, including at least one from each orchard (Np1, Np2, Np3), were selected for further analysis. Genomic DNA was extracted using Axygen MAG-FRAG-I-50 (Axygen Bio-Tek). The internal transcribed spacer of rDNAs (ITS), beta-tubulin (tub2), the nuclear ribosomal large subunit (LSU), and translation elongation factor 1-α (tef1-α) gene regions were amplified using the primers ITS1/4, Bt2a/Bt2b, LR5/LR0R, and EF1-728F/EF1-986R (Golzar and Burgess 2011) and sequenced. Sequence analysis using MEGA 7.0 (Kumar et al., 2018) revealed 100% similarity among the isolates. BLAST searches of the ITS, tub2, LSU, and tef1-α sequences (accession nos. MN907637, MT537938, MT528156, MT537939) showed the highest nucleotide similarities (99.24 to 100%) to Neofusicoccum parvum strains (Crous et al. 2006). A phylogenetic tree was constructed with MEGA 7.0 based on the nucleotide sequence tub2 using the maximum likelihood method. Pathogenicity tests on 10 healthy guava fruits using mycelium-inoculated and control fruits confirmed the causative agent. The inoculated fruits, maintained at 25°C under a 12-h light/dark cycle, exhibited symptoms identical to the field infections within four to seven days, while control fruits remained symptomless. The fungus, reisolated from the inoculated fruits, was morphologically identical to the original isolates, fulfilling Koch's postulates. In conclusion, based on molecular, morphological, and pathogenic analysis, N. parvum as the causal agent of the fruit rot disease on guava. Previously, N. parvum has been reported in association with fruit rot on Eriobotrya japonica and Juglans regia (Zhai et al. 2019; Chen et al. 2019). To our knowledge, this is the first report of N. parvum affecting guava in China.

First Report of Bacterial Leaf Streak of Rice Caused by Pantoea ananatis in Guangdong Province, China.

Rice (Oryza sativa L.) is a crucial staple crop worldwide, and bacterial diseases are among the primary factors affecting rice yield. In late October 2022, bacterial leaf streak disease was observed on the leaves of the rice variety Meixiangzhan 2 across multiple fields (approximately 130 hm2) in Leizhou City, Guangdong Province, China. The incidence rate was up to 30% in each field. Infected rice leaves exhibited distinctive symptoms at the boundary between diseased and healthy tissue, featuring dark green to yellow-brown streaks, while most of the leaf margin exhibited symptoms of either leaf edge or sheath rot. Disease progression from the leaf tip inwards revealed gray-white or dehydrated lesions with a bluish-gray color. Some leaves exhibited wrinkling at the edges, and severe symptoms at the leaf tip resembled those of bacterial leaf blight in rice. Ten leaves were collected from 10 infected rice plants in three distinct fields, and leaf pieces at the border of diseased and healthy areas were surface disinfected with 75% anhydrous ethanol for 60 seconds, rinsed three times with sterile water, and then soaked in sterile water for 8 hours. The obtained bacterial suspension was diluted at a ratio of 1: 106, and 100 μL of the diluted samples were plated on Potato Dextrose Agar (PDA) plates. After incubation at 28°C for 48 hours, the yellow bacterial colonies that appeared, were purified on PDA plates. To confirm the bacterial species, the amplification of genes gyrB, leuS, rpoB, and 16S rDNA was performed on six randomly selected isolates from the three different fields using the primers 27F/1492R, gyrB-F/R, leuS-F/R and rpoB-F/R, as reported by Yu et al (2022), respectively. PCR products were sequenced. All six isolates had identical sequences for all genes sequenced.The gene sequences of 16S rDNA (960 bp), gyrB (953 bp), leuS (733 bp), and rpoB (877 bp) for LZ1, were deposited in the NCBI database under accession numbers PP048830 , PP068625 , PP068626, and PP068627, respectively. These sequences were subsequently compared using BLASTn tool against the NCBI nr/nt database. The 16S rDNA, gyrB, leuS, and rpoB of LZ1 showed similarities of 99.90%, 99.16%, 99.73%, and 99.89%, with the corresponding sequences of P. ananatis TZ39 (GenBank accession numbers MZ800600.1 for 16S rDNA, and CP081342.1 for gyrB, leuS and rpoB ). MLSA analysis using concatenated sequences of gyrB, leuS, and rpoB genes indicated that the isolated strain LZ1 belongs to P. ananatis. In the tillering stage of rice varieties Meixiangzhan 2 and Huahangyuzhan, P. ananatis LZ1 was inoculated at a concentration of 108 CFU/mL using the leaf-cutting method, with sterile water used as a control (Toh et al., 2019). After 14 days of bacterial inoculation, the inoculated leaves gradually became necrotic, changing from light green to brown showing identical symptoms as those in the field, while the control plants remained symptom-free. Subsequent 16S rDNA, gyrB, leuS and rpoB gene sequencing results further confirmed the identity of the pathogen as P. ananatis, thereby fulfilling Koch's postulates. Previous reports have already identified P. ananatis as the pathogen causing rice bacterial leaf streak (Kini et al., 2017; Arayaskul et al., 2019; Yu et al., 2022; Lu et al., 2022; Luna et al., 2023; Yuan et al., 2023). This is the first report of rice bacterial leaf streak caused by P. ananatis in Guangdong Province, China, laying the foundation for future research to establish strategies for the prevention and control of this disease.

First report of anthracnose caused by Colletotrichum grevilleae on apple in Korea.

In October 2022, typical symptoms of anthracnose were observed on apple (Malus ⅹ domestica cv. Fuji) fruits collected from Pocheon in Gyeonggi province, South Korea (N37.98074°, E127.33995°). In the surveyed orchard, the incidence rate of apple anthracnose was less than 1%. The initial symptoms were brown-to-dark brown lesions, and with disease progression, they enlarged and the pulp became soft, forming a brown band. In total 29 apple fruits were collected, and the causal agent was isolated by removing the peel, and the diseased tissues were directly transferred onto potato dextrose agar (PDA), followed by incubation for 7 days at 25°C. As the results, two isolates (GgPc22-1-11 and GgPc22-1-13) were obtained. For describing morphological and cultural characteristics, isolate GgPc22-1-11 was cultured on PDA and synthetic nutrient-poor agar (SNA) at 25°C under near-UV light with a 12-h photoperiod for 10 days. The colonies of GgPc22-1-11 on PDA were initially white and subsequently appeared light gray to olivaceous with white margins. The reverse side of the plates were dark brown and slate blue (Supplementary Fig. S1). Colonies on SNA were flat with an entire margin and short sparse white aerial mycelium. No setae were observed. Conidia on PDA were hyaline, straight, aseptate with a rounded apex, clavate to cylindrical, and measured 16.4 ± 2.4 (10.8-23.8) × 5.5 ± 0.7 (3.6-7.7) μm (n = 200). Appressoria were medium-to-dark brown, aseptate, solitary or in groups with irregular outlines, and lobate or having undulate margins (Supplementary Fig. S1). These morphological and cultural characteristics of GgPc22-1-11 were consistent with those of Colletotrichum grevilleae F. Liu, Damm, L. Cai & Crous, pathogens of Proteaceae and Punica granatum (Liu et al. 2013; Huang et al. 2023). DNA was extracted from GgPc22-1-11, PCR was performed and Phylogenetic analysis of concatenated partial sequences of the internal transcribed spacer (ITS) of rDNA, β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and actin (ACT) genes was conducted (Weir et al. 2012). The resulting sequences were deposited in GenBank under the accession numbers LC773710-LC773714. A nucleotide BLAST search revealed that the ITS sequences of the isolates were 98.95% identical to those of C. grossum CAUG7 (KP890165.1). The TUB2, GAPDH, CHS-1, and ACT sequences of the isolates were 99.79%, 99.24%, 100%, and 100%, respectively, identical to those of C. grevilleae WP4. GgPc22-1-11 was clustered with C. grevilleae WP4 using neighbor joining analysis conducted with MEGA X software (Kumar et al. 2018) (Supplementary Fig. S2). Pathogenicity tests were conducted using GgPc22-1-11 and repeated three times. A total of 12 symptomless apples of each variety were selected, including Fuji, Hongro, Tsugaru, and RubyS. The apples were surface-sterilized with 70% ethanol and wounded using a sterile needle. Both wounded and unwounded apples were inoculated with mycelium plugs and paper disks containing a conidial suspension (1 × 106 conidia/ml) and placed in a plastic box with moist paper towels (>90% relative humidity) at 25°C in dark. At 5 days after inoculation, all artificially wounded fruits exhibited symptoms and 30% (4 out of 12) of unwounded inoculated fruits showed symptoms in each apple variety while control fruits were asymptomatic both the unwounded and wounded inoculations (Supplementary Fig. S1). To fulfill Koch's postulates, the fungi were reisolated from symptomatic tissues and were identical to GgPc22-1-11 confirmed by morphological and molecular analysis. To the best of our knowledge, C. grevilleae has been reported in Protea sp. and pomegranate (Liu et al. 2013; Huang et al. 2023) but not in apples to date, and this is the first report of C. grevilleae causing anthracnose in apple fruits. This research of the newly emerged unreported Colletotrichum species can offer valuable information for development of an effective fungicide spray program to control apple anthracnose.

First report of Sclerotium hydrophilum causing leaf spot on Brasenia schreberi in central China.

Brasenia schreberi, commonly known as watershield and referred to as 'Chun Cai' in Chinese, is a worldwide aquatic vegetable. It has long been regarded as health- promoting vegetable due to production of mucilage in young shoots, and thus has gained popularity in China. In September 2022, a leaf spot disease was observed at the National Aquatic Vegetable Germplasm Resource Nursery located in Wuhan, Hubei province, China. The disease occurred on watershield leaves. It started with the formation of leaf spots surrounded by halos.These spots ranged in color from yellow to brown and in diameter from 1 to 10 mm. Subsequently, the smaller spots merged, ultimately causing the entire leaves to turn black. Small brown- to black-colored sclerotia were produced on the underside of the diseased leaves. Disease incidence was 30% on average, and yield loss was approximately 15% on average. To isolate the pathogen, leaf tissues at the disease-healthy border area were excised into 5 × 5 mm pieces, these segments were immersed in 75% ethanol for 30 s, followed by immersion in 0.5% sodium hypochlorite for 30 s, and then rinsed twice in sterile water. After air-drying, the leaf pieces were incubated on potato dextrose agar (PDA) in darkness at 28°C for 3 d. Mycelia from the leaf pieces were transferred to new PDA plates for purification, three sclerotia-forming fungal isolates (Whcc-1, Whcc-3, Whcc-4) were finally obtained. They were incubated on PDA at 28°C for 4 to 14 d for observation of colony morphology. At 4 d after incubation (DAI), they grew rapidly with the average growth rate of 2.2 cm/d and formed colonies with whitish substrate mycelia and well-developed aerial mycelia and small white to light brown-colored sclerotia. At 10 to 14 DAI, the sclerotia gradually turned to black, 0.2 to 0.4 mm in diameter (0.26 mm on average, n = 50). These morphological characteristics matched description of Sclerotium hydrophilum (Bashyal et al. 2021). Molecular identification was done to further clarify the species identity of this fungus. Genomic DNA was extracted from isolates Whcc-1, Whcc-3, and Whcc-4. The internal transcribed spacer region of the ribosomal DNA (ITS-5.8S rDNA) and the small subunit ribosomal RNA gene (ssrRNA) were amplified using universal primers ITS1/ITS4 and NS1/NS6, as described by White et al. (1990). Sequence analysis revealed a high degree of similarity between the ITS-5.8S rDNA sequences from Whcc-1, Whcc-3, and Whcc-4 (GenBank Acc. No. OP782030, PP035994, and PP035995, respectively) and those of S. hydrophilum strain CBS201.27 (GenBank Acc. No. FJ231396), with similarities of 99.25%, 99.4%, and 99.25%, respectively. The ssrRNA sequences from Whcc-1, Whcc-3, and Whcc-4 (GenBank Acc. No. PP238401, PP261342 and PP261345) were found to be identical, displaying 100% similarity to the ssrRNA sequences of S. hydrophilum strain ZH11 (GenBank Acc. No. KC354147). Based on both morphological and molecular evidence, it can be inferred that Whcc-1, Whcc-3, and Whcc-4 belong to the species S. hydrophilum. Pathogenicity tests were conducted by inoculation of the mycelial agar plugs of Whcc-1, Whcc-3 or agar plugs of fresh PDA (control) on floating leaves of two watershield plants, 4 leaves (replicates) for each treatment. After inoculation, the treated leaves were sealed in plastic bags to maintain humidity, and grown under natural conditions (18°C to 28°C, with 8 hours of light daily). At 7 DAI, while control leaves remained healthy, the leaves inoculated with Whcc-1 and Whcc-3 leaves formed yellow- to brown-colored spots similar to those observed in the field surveys. S. hydrophilum was re-isolated from the leaf spots, thus verifing Koch's postulates. S. hydrophilum has a wide host range, infecting at least 19 genera of plants, including common rice and wild rice (Johnson et al. 1976), and water lily (Kernkamp et al. 1977). Moreover, it has been reported to infect rice in China (Punter et al. 1984; Zhong et al. 2018). To our knowledge, this is the first report of S. hydrophilum on watershield leaves in central China.

First Report of Anthracnose on Euonymus japonicus Thunb. Caused by a Provisionally Novel Species of Colletotrichum in the Magnum Complex in South Korea.

Euonymus japonicus Thunb., also known as the evergreen spindle tree, is an evergreen tree, which is widely planted as a hedge plant along streets in South Korea. In April 2022, severe anthracnose symptoms were observed on the leaves of this tree in Jangsu in the Jeonbuk Province of the country (35°43'49.44″N, 127°34'53.7″E). About 80% of the leaves of each affected tree within a 0.03-ha area showed incidence of the disease on approximately 30 trees were planted along the roadside (~30 m). These symptoms typically included circular or irregularly shaped whitish-gray lesions with a diameter of 2.0 to 3.0 cm. In cases where some leaves were severely affected, larger blotches formed. To isolate the pathogen, about ten leaves showing anthracnose symptoms on each tree were randomly selected and brought to the laboratory. Fungal isolations were made from acervuli filled with conidial masses on infected evergreen tissues, followed by plating onto 2% potato dextrose agar (PDA) as well as incubated at 25℃. On the PDA, colonies were circular, raised, green-grey or dark grey, and had a distinct white margin. The conidia were single-celled, transparent, cylindrical with rounded ends, had smooth walls, with a length ranging from 12 μm to 16.7 μm and a width raging from 4 μm to 6.5 μm (av. = 14.1 X 5.0 μm, n=40). Of those that were successfully recovered with approximately 90% frequency, two monoconidial isolates were deposited to the culture collection at Chungnam National University in South Korea (Accession number: CDH059-060). To ensure the identity of the fungus, genomic DNAs were extracted from the selected isolates, CDH059-060, and were sequenced. This was achieved based on partial sequences of the internal transcribed spacer (ITS), actin and beta-tubulin (TUB2) gene regions which were amplified using ITS1F / ITS4 (Gardes and Bruns 1993; White et al. 1990), ACT-512F / ACT-783R (Carbone and Kohn 1999), and T1 / Bt2b (O'Donnell and Cigelnik 1997; Glass and Donaldson 1995) primer pairs, respectively. The resulting sequences were deposited to GenBank (OR984424-425) for ITS, (OR996289-290) for actin, and (OR996291-292) for TUB2. For a phylogenetic analysis, sequences from different gene regions (ITS, actin and TUB2) retrieved from GenBank were aligned, concatenated, and analyzed as a single dataset based on a maximum likelihood analysis. The phylogenetic result revealed that the fungus isolated in this study was positioned in a clearly distinct lineage, provisionally representing an undetermined species of Colletotrichum, which is most closely related to Colletotrichum liaoningense (Y.Z. Diao, C. Zhang, L. Cai & X.L. Liu, CGMCC3.17616 (KP890104 for ITS, KP890097 for actin, and KP890111 for TUB, Diao et al. 2017). Sequence comparisons revealed that this pathogen differed from C. liaoningense at 20 of 494 characters (∼4.0%) in the ITS and 2 of 251 (∼1.0%) in the actin sequences. For pathogenicity tests, three seedlings of E. japonicus were used. The leaves for each tree were treated with 10 ml of a conidial suspension by spraying (1x10⁶ conidia ml-1 of the isolate, CDH059), while the three seedlings were treated with distilled water as control. After sprayed, the treated areas were sealed with plastic bags for a day to maintain humidity. Anthracnose symptoms identical to those observed in the field appeared seven days after inoculations, while no symptoms were observed in the control. Re-isolations were successfully achieved from the treatments, fulfilling Koch's postulates. Anthracnose associated with the provisionally novel species of Colletotrichum sp. on E. japonicus has not been recorded elsewhere, and in this regard, this is the first report of anthracnose caused by Colletotrichum sp. on E. japonicus in Korea. To effectively control the disease, more attention should be paid to the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

First report of leaf blight caused by Stemphylium lycopersici on Salvia splendens in China.

Salvia splendens is a popular ornamental plant in China with extensive potentials, including value in traditional Chinese medicine and in environmental restoration function (Li et al. 2008). In September 2019, leaf blight disease was observed on road side plants of S. splendens in Bayi park, Nanchang city, Jiangxi province, China. The typical symptoms appeared as irregular necrotic spots or leaf blight, accompanied by extensive scorch necrosis or ultimately defoliation. Small segments cut from diseased leaves were surface sterilized in a 2% sodium hypochlorite solution for 2 min and rinsed three times with sterile distilled water. Then, the samples were placed on potato dextrose agar (PDA) plates incubated at 25°C in darkness. Pure cultures were obtained by the hyphal tip method. Morphologically, all 11 colonies were identical to each other on PDA. Two strains, YZU 191468 and YZU 191481, were selected for further study and deposited in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. The 7-day-old colonies were circular, 53 to 56 mm in diameter, and consisted of white mycelium with a buff margin, and were cinnamon colored in the center of the reverse side. To examine conidial morphology, the mycelium was transferred onto potato carrot agar (PCA) and incubated at 23°C with a period of 8 h light/16 h dark for 7 days. Conidia were normally solitary or two in a chain, ellipsoid or long ellipsoid, beakless, 10 to 23×30 to 60 µm in size (n=50). Based on morphology, the isolates were consistent with Stemphylium lycopersici (Yamamoto 1960). To confirm the identification, genomic DNA was extracted from both isolates and used to amplify the internal transcribed spacer rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and calmodulin (CAL) genes with primer pairs ITS5/ITS4, gpd1/gpd2, and CALDF1/CALDR2, respectively (Woudenberg et al. 2017). Sequences were deposited in GenBank with accession numbers OP564983 and OP564984 (ITS), OP892529 and OP892530 (GAPDH), OP584970 and OP584971 (CAL). A neighbor-joining tree was constructed with Mega 7.0 based on the combined dataset with 1,000 bootstrap replicates. The resulting phylogenetic tree showed that the strains from S. splendens clustered with S. lycopersici (CBS 122639 and CBS 124980) supported with 100% bootstrap values. The molecular analyses confirmed that the species causing leaf blight symptoms was S. lycopersici. To test pathogenicity, healthy leaves of S. splendens were surface sterilized and inoculated by mycelium blocks (6 mm in diameter) and spore suspension (1×106 spore/mL) of representative strains YZU 191468 and YZU 191481, respectively. Controls were inoculated with blocks of PDA and sterile water. Each strain was inoculated on three leaves of a plant. One clean plant was used as control. The test was replicated three times. After inoculation, the plants were covered with plastic bags and incubated in a greenhouse (25℃, 80 % relative humidity, 8 h light/16 h dark). After 5 days, the inoculated leaves exhibited dark brown spots with white mycelium, followed by withering of necrotic tissues. There were no symptoms observed on the controls. The fungal isolates inoculated leaves had the same morphological characteristics as the strains used for inoculation. S. lycopersici has been found on eggplant and Zinnia elegans in China (He et al. 2019; Yang et al. 2017). To the best of our knowledge, this is the first report of S. lycopersici causing leaf blight on S. splendens in China. This finding offers a new reference for the management and control of S. splendens leaf diseases in China.

First Report of Brown Leaf Rot Disease on Dendrobium nobile Caused by Xylaria flabelliformis in Guizhou Province, China.

Dendrobium nobile is the largest species of the Orchidaceae family and produces dendrobine, a compound with medicinal properties (Sarsaiya et al., 2020a; Sarsaiya et al., 2024; Qian et al., 2024). The accumulation of dendrobine in D. nobile is regulated by various pathogenic fungi, which directly and indirectly influence dendrobine biosynthesis (Sarsaiya et al., 2019a; Sarsaiya et al., 2019b). In a field planted with D. nobile in Guizhou Province, China, small lesions were initially observed on the upper part of the leaves from May to June 2019, which later developed into larger brown necrotic leaf lesions. Over time, these lesions greatly impacted the medicinal value (dendrobine) and productivity of the plant. A pure culture of Xylaria flabelliformis from infected wild D. nobile leaves was recovered and subsequently cultured on potato dextrose agar (PDA) at 25 °C for 5 days. Xylaria flabelliformis grew slowly and was composed of white mycelia. Colonies were initially white, with a regular margin, and formed stromata that consisted of mycelia sterilia without ascospores. We identified the strain as Xylaria flabelliformis based on its morphological characteristics (Liu et al., 2007) and by sequencing elongation factor-1α (EF-1α). The length of the DNA sequence of EF-1α that was used for the analysis of Xylaria flabelliformis was 1188 bp. BLASTx (nucleotide 6-frame translation-protein) analysis using the National Center for Biotechnology Information database showed that the obtained protein sequence (BLASTx protein accession no.: UTS95822.1, BLASTn nucleotide sequence accession no.: MW508334.1) had the highest similarity (98.21%) with the X. flabelliformis hypothetical protein (TRX95197.1) based on a thorough phylogenetic comparison with other Xylaria species. Healthy D. nobile seedlings were planted in pots and sterilized. The terminal leaves were excised from all pre-sterilised D. nobile seedlings and inoculated with Xylaria flabelliformis mycelial plugs, whereas sterile PDA plugs and moist cotton plugs were used as controls. All seedlings were maintained under optimum temperature and humidity conditions (25 °C and 80%, respectively) for seven days for observation and analysis. All experiments were performed in triplicate. After the incubation period, brown leaf rot lesions were observed for the first time on the inoculated D. nobile leaves, but no symptoms were observed on the leaves of the two control groups (sterile PDA plugs and moist cotton plugs). To complete Koch's postulates, Xylaria flabelliformis was re-isolated and identified from all diseased tissues by DNA sequencing of the EF-1α. It was determined for the first time that Xylaria flabelliformis can cause brown leaf lesions in D. nobile. Moreover, the pathogenicity of Xylaria flabelliformis in D. nobile has not been previously reported (Mead et al., 2019; Meng et al., 2019; Sarsaiya et al., 2019a; Sarsaiya et al., 2020b; Chen et al., 2023; Rinchen, 2023; Cao et al., 2024). To the best of our knowledge, this is the first report of BLRS lesions in D. nobile leaves caused by Xylaria flabelliformis in Guizhou Province, China. Identification of Xylaria flabelliformis as a pathogen of D. nobile is crucial for advancing effective management and control practices against brown leaf rot disease. This discovery provides valuable insights into the development of targeted strategies to mitigate the impact of Xylaria flabelliformis on D. nobile, safeguard medicinal properties such as dendrobine, and enhance overall productivity.

Influence of Foliar Treatment with Suspensions Rich in Trichoderma Chlamydospores on Momordica charantia Physiology, Yield, and Quality

Several strategies promote phyllosphere colonization by soil-born Trichoderma plant-beneficial strains. One of these strategies is foliar spraying with suspensions containing large amounts of chlamydospores—spores with thick cell wall structures that make them highly resistant to harsh environmental conditions. Trichoderma biomass was produced by cultivation on a cornmeal medium and compared with the biomass produced on potato dextrose broth by microscopic and thermogravimetric analyses. The analyses revealed increased chlamydospore content and thermostability in the fungal biomass produced on the corn meal medium. The Trichoderma suspension rich in chlamydospores was sprayed on bitter gourd (Momordica charantia) leaves at two inoculant concentrations, 106 and 108 ufc/mL. The effect of these treatments on the plant physiological parameters, leaf photosynthetic pigments, polyphenol and flavonoid contents, antioxidant activities of the leaves and fruits, and yield was compared to the control (plants sprayed with water) and to the experimental treatment involving spraying with 108 ufc/mL of propagules produced in potato dextrose broth. The effect of chlamydospore-rich suspensions on plant physiological parameters was more pronounced and long-lasting compared with the other treatments. The treatment with chlamydospore-rich suspension enhanced the accumulation of polyphenols and flavonoids in the leaves (by 17% and 50%, respectively) and fruits (by 18% and 31%, respectively) and increased the antioxidant activity. The Trichoderma treatment increased the yield by +25.33–53.07%. The application of the foliar treatment with Trichoderma suspensions did not modify the cytocompatibility of the extracts from the fruits determined on the L929 cells.

First Report of Shoot Blight of Grapevine Caused by Sclerotinia sclerotiorum in Illes Balears, Mallorca, Spain.

In 2021, grapevines (Vitis vinifera L.) cv. Callet growing in a commercial vineyard located at Pollença (northeast of the island of Majorca, Spain) showed severe symptoms of shoot blight during spring and early summer, with an incidence of 70%. Symptoms consisted of elongated cankered-like lesions, surrounded by water-soaked darker tissues, that developed at the base or around the middle nodes of the shoot. For fungal isolation, shoot samples with lesions were collected, surface disinfected with 2% NaCl for 90s, rinsed twice with deionized water and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25°C under 12 h light-darkness for 6 days. Isolations consistently yielded on kind of fungal colonies that produced white mycelium and black spherical to elongated sclerotia (2 to 10 mm in diameter). Morphological characterization was consistent with the description of Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006). Three isolates (UIB 118-1, UIB 118-26, and UIB 129-41) were preserved and deposited in the Culture Collection of Microbiology-Faculty of Sciences, University of Balearic Islands, Spain. Genomic DNA was extracted from isolates UIB 118-26 and UIB 129-41 using the EZNA Miniprep Kit (Omega Bio-Tek, Norcross, GA). The internal transcribed spacer (ITS) region of ribosomal DNA, β-tubulin (BTUB) and calmodulin (CAL) gene regions were amplified using ITS1F-ITS4 (Gardes and Bruns, 1996; White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995) and CAL228F/CAL737R (Carbone and Kohn 1999) primer sets, respectively. Amplicons were sequenced and deposited in GenBank with accession numbers MZ604647 and MZ604648 for ITS, OK634402 and OK634403 for BTUB and OK634404 and OK634405 for CAL. BLASTn search showed that isolates were >99 % (ITS, BTUB and CAL) identical to S. sclerotiorum GenBank accession no. KF859933, CP017815 and KF871381, respectively. Pathogenicity tests were conducted using eight one-year old grapevines cv. Cabernet Sauvignon. Old and new green shoots were inoculated by inserting a 6-mm plug of mycelium taken from actively growing cultures on PDA into cuts made at the base and at the distal part of each shoot with a sterile scalpel with a total of eight inoculation points per plant. Inoculated wounds were sealed with Parafilm tape to avoid rapid dehydration. Inoculated plants and an equal number of wounded but non-inoculated plants (negative controls) were maintained at 25 ± 1°C for 48 h in plastic containers to ensure a high relative humidity (>90%). After 5 days, the infection girdled and rotted the green new shoots, whereas the older partially lignified shoots developed a localized long brown lesion that reached 16 cm in length. Due to the rotting of the basal part of the petiole, leaves turned gray, wilted, and died, easily detaching from the stem. In advanced stages of the disease, 7 days after infection, branches died and fell with the leaves remained attached (Fig 1 A, B). Reisolations from diseased shoots were successfully performed on PDA to fulfill Koch's postulates. S. slerotiorum was previously reported on grapevine causing shoot blight in Chile (Latorre and Guerrero, 2001), Korea (Jong-Han et al. 2009), California-USA (Boland and Hall, 1994) and Australia (Hall et al. 2002). AlsoS. sclerotiorum was reported among the endophytic mycobiota associated with Vitis vinifera in the Iberian Peninsula (Gonzalez and Tello, 2011) but not as a pathogen causing visible symptoms on that crop. So, this is the first report of the occurrence of S. slerotiorum as a pathogen of grapevines in Spain causing symptoms of canker and shoot blight. This finding highlights a potential risk of this fungal disease for the wine industry in the Mediterranean region and specially for Spain, the country with the largest acreage devoted to grapevines. Although chemical and biological are suitable control strategies, disease management is difficult as sclerotia of Sclerotinia can remain in the soil for up to eight years (Adams and Ayears, 1979), and preventive surveys are greatly recommended as an important epidemiological tool to monitor the epidemiology of disease and identify potential outbreaks of this new pathogen on grapevine in Spain.

Evaluation of Antifungal Activity of Endophytic Bacillus spp. and Identification of Secondary Metabolites Produced Against the Phytopathogenic Fungi.

Endophytic bacteria serve as a rich source of diverse antimicrobial compounds. Recently, there has been a growing interest in utilizing endophytic Bacillus spp. as biological agents against phytogenic fungi, owing to their potential to produce a wide range of antimicrobial substances. The objective of this research was to investigate the protective abilities of 15 endophytic Bacillus spp. isolated from previous study from wheat plant, against the phytopathogenic fungi, Fusarium graminearum and Macrophomina phaseolina. A dual culture plate assay was conducted as a preliminary analysis, revealing that 7 out of 15 endophytic Bacillus spp. demonstrated inhibition against one or both of the phytopathogenic fungi used in this study. All seven endophytes were further assessed for the presence of diffusible antifungal metabolites. The cultures were grown in potato dextrose broth for 120h, and the cell-free supernatant was extracted and analyzed using the cup plate method. The methanolic extract yielded similar results to the dual culture plate analysis, except for WL2-15. Additionally, deformities in the mycelial structure were examined under the light microscope upon exposure to methanolic extract. Furthermore, the analysis and identification of metabolites were carried out via gas chromatography-mass spectrometry of methanolic extract from selected seven endophytic Bacillus spp. The chromatogram revealed the presence of some major peaks such as tridecanoic acid, methyl ester, hydroperoxide, 1-methylbutyl, 9-octadecenamide, (z)-, hexane-1,3,4-triol, 3,5-dimethyl- tetradecanoic acid. To the best of our knowledge, this is the first report of these biocontrol agents in endophytic Bacillus spp. Interestingly, volatile organic compound production was also seen in all the isolates against the phytopathogenic fungi.