White Mycelium Research Articles

First record of Aspergillus caelatus as a pathogen of Heterotrigona itama and Geniotrigona thoracica in Malaysia

Heterotrigona itama and Geniotrigona thoracica become popular in Malaysia as honey producer in the 21st century. Like other bee species, stingless bees can be infected by pathogens. Disease-related population declines may affect honey production, agricultural productivity, and biodiversity. Thus, this study aimed to evaluate the health status of stingless bee colonies in selected bee farms in Malaysia. Adults Heterotrigona itama and Geniotrigona thoracica were collected from 79 colonies in 9 farms and screened for known pathogens of honey bees and stingless bees using 15 primer sets. All results were negative, indicating the absence of these pathogens in the bee specimens. During the bee sampling at Infoport Halal Hub UPM, we encountered a collapsed colony of H. itama. A fungal isolate was isolated and confirmed to be Aspergillus caelatus. Virulence tests revealed that A. caelatus exhibited high virulence to both H. itama and G. thoracica. Infected larvae became lethargic after 1–2 day after treatment (DAT) with white mycelium developed on their integument. The fungus caused 100 % larval mortality in both H. itama and G. thoracica after 8 DAT, with LC50 values of 7.58 × 103 conidia/µl and 6.24 × 103 conidia/µl respectively. Yellow-green conidia were observed on the cadavers. A. caelatus demonstrated high virulence towards both H. itama and G. thoracica, leading to severe larval mortality. This study represents the first documented case of a disease infecting stingless bees in Malaysia. Future research is needed to assess the prevalence of A. caelatus in various stingless bee species and other pollinators within the country.

First report of powdery mildew on Euonymus japonicus caused by Erysiphe euonymicola in Taiwan.

The Japanese spindle (Euonymus japonicus Thunb.) is commonly used as an ornamental hedge plant in Taiwan. In March 2020, a severe powdery mildew disease was observed on E. japonicus surrounding a city park spanning six hectares in Taichung city, Taiwan. Around 90% of the plants showed symptoms on the leaves and pedicels of young shoots. Similar symptoms were observed in other districts of Taichung city and Taipei city between March to June in subsequent years. Initial signs of infection manifest as circular chlorotic spots on the leaves, which are subsequently covered by white mycelia on either the upper or lower surfaces of the spots. In severe cases, both sides of the leaves become entirely covered by dense mycelia. Hyphal appressoria were solitary or in opposite paired, lobed to multilobed. Conidiophores grow erectly from the hyphae, consist of 2-3 cylindrical cells, 38.9 to 78.6 × 6.31 to 8.28 µm (n = 30). Foot cells are usually straight or slightly flexuous, 23.6 to 43.2 µm (n = 30), followed by 1 to 2 shorter cells. Ellipsoidal conidia are produced singly on the conidiophores, 24.1 to 36.3 × 10.6 to 14.97 µm (n = 30), without fibrosin bodies. Germ tubes are mostly subterminal, sometimes terminal, occasionally exhibiting a longitudinal pattern. Chasmothecia were not observed. These morphological characteristics correspond to the description of Erysiphe euonymicola U. Braun (Braun and Cook 2012), one of the Erysiphe species reported on E. japonicus. Genomic DNA was extracted from seven isolates obtained from different plants in the affected regions. The internal transcribed spacer (ITS) and 28S large subunit (LSU) of rDNA sequences (ITS accession nos.: OR073423-OR073429; LSU accession nos.: OR073448-OR073454) were amplified and sequenced using primer sets PMITS-1 / PMITS-2 (Cunnington et al. 2003) and NLP2 / PRM2 (Bradshaw and Tobin 2020), respectively. The resulting sequences exhibited identities ranging from 99.1 to 100% in ITS and 100% in LSU when compared to the corresponding sequences of E. euonymicola MUMH 133 (ITS: AB250228; LSU: AB250230) (Limkaisang et al. 2006). Phylogenetic analysis based on the concatenated sequences of ITS and LSU clustered the seven isolates within the same clade as three E. euonymicola isolates (MUMH 133, MUMH 6999 and MUMH 7012). Pathogenicity assays were conducted on one-meter tall E. japonicus plants by gently smearing infected leaves on all leaves of four healthy plants. Four uninoculated plants were used as control. All eight assayed plants were enclosed in plastic bags to maintain high humidity at 28 ± 2°C for 3 days. Chlorotic spots began to appear on leaves younger than one month old at 7 days post inoculation (dpi). By 28 dpi, all inoculated plants showed symptoms. Spots expanded or merged and formed a dense mycelial layer on leaves younger than three months, while mature dark green leaves were asymptomatic. No symptoms were observed on any leaves of the control plants. The morphological characteristics and sequences of ITS and LSU of the pathogen from the inoculated plants matched the above information. Based on these findings, E. euonymicola was identified as the causal agent of powdery mildew on E. japonicus, representing the first documented report of this disease in Taiwan. A voucher specimen TNM F0037001 (isolate EPM-1) was deposited in the National Museum of Natural Science, Taiwan. The pathogen has been frequently reported in recent years and significantly impacts the ornamental value of Euonymus spp. (Abbasi and Braun 2020; Lee et al. 2015; Li et al. 2011; Pei et al. 2022). This report also provides an evidence of an ongoing outbreak of the pathogen.

Identification of stem rot of stock (Matthiola incana) caused by Sclerotinia sclerotiorum in China

The ornamental plant stock flower (Matthiola incana (L.) R. Br.) has been widely cultivated in China. In 2021, stem rot on M. incana, which caused plant death, was first observed in Shanghai, China. Field surveys indicated a disease incidence of 15%–20%. Following pathogen isolation, identification was performed by combining morphological observation of colonies, sclerotia and apothecia, with molecular methods, including DNA sequencing and phylogenetic analysis. Morphological characteristics of the isolates, including white cottony mycelium, blackish globular to irregularly shaped sclerotia and apothecia formation, were consistent with Sclerotinia sclerotiorum. Results of sequence comparison and phylogenetic analysis based on the internal transcribed spacer (ITS) sequences supported morphological data. Pathogenicity assays were conducted successfully according to Koch's postulates. Considering the results of the morphological and molecular identification, along with the pathogenicity assessment, S. sclerotiorum was confirmed as the causal agent of stem rot of stock flower in Shanghai, China. A combination of cultural practices, rotation of resistant ornamentals, and biological and chemical treatments is recommended to control stem rot of stock.

First report of powdery mildew caused by Erysiphe corylacearum on hazelnut in Bulgaria.

In June 2020, during an observation of fruit-bearing hazelnut trees (Corylus avelana L., cv. Tonda Gentile), grown in a small private home garden in the region of Kavarna (North-Eastern Black Sea coast of Bulgaria), symptoms of powdery mildew were observed on each of the 15 trees. Compared to the widely spread in Bulgaria powdery mildew caused by Phyllactinia guttata and known with its powdery patches and differing chasmothecia exclusively formed on the abaxial leaf surface, the newly observed disease was characterized by the presence of concomitant pathogen's structures on both the abaxial and adaxial surfaces of the leaves, varying from single pustules to 90% severity on certain leaves, as well as on the husks which were 100% affected. Among the white mycelium, cylindrical conidiophores and singly produced conidia (22-34 x 16-22 µm; n = 50) were found. Scattered or in groups, black spherical chasmothecia (diameter of 85-115 µm; n = 30), possessing 8 to 12 appendages with dichotomous branched apices, were also observed in the autumn. Chasmothecia contained 3 to 5 subglobose asci (45-60 x 34-62 µm; n = 30), each ascus containing 8 hyaline and ellipsoid ascospores (15-22 x 9-16 µm; n = 50). From one field powdery mildew sample DNA was extracted using the Nucleospin Plant II kit (Macherey-Nagel, Germany) and the rDNA ITS region was amplified using primers PMITS1 and PMITS2 (Cunnington, J.H. et al., 2003). The amplicon was sequenced (Macrogen Europe, The Netherlands) and used in a BLAST analysis. This resulted in 100% homology with the ITS sequence of Erysiphe corylacearum. The obtained sequence was different from the ones of Erysiphe pseudocorylacearum (99.20% homology) and Erysiphe coryli-americanae (96.52% homology). The targeted sequence was submitted to GenBank (accession number OQ860748). Based on the symptoms, morphological characteristics and molecular identification, the pathogen was identified as Erysiphe corylacearum. Pathogenicity of the fungus was confirmed under lab conditions (natural day/light regime, 23-25°C) by shaking down conidia from naturally infected leaves on both sides of healthy leaves (2-years plantlets, cv. Tonda Gentile, 3 replicates) and incubation in a moist chamber (plastic bags) for the first 24 hours. First small fluffy white pustules appeared 6-7 days after inoculation, subsequently, the disease was progressing and the pathogen's conidiophores and conidia were morphologically identical to that first observed under natural infections in the private garden. The non-inoculated control plantlets (grown under the same conditions in a separate room) remained symptomless during the entire three weeks testing period. To our knowledge, this is the first report of Erysiphe corylacearum on hazelnut in Bulgaria. In October 2021, the same disease was also found in a hazelnut orchard (1.2 ha) located in the region of Pazardzhik (Central Southern part of the country). Globally, this report indicates the disease's expansion in the Black Sea region since its first observation in Turkey in 2013 (Sezer, A., et al. 2017; Heluta, V.P., et al. 2019; Meparishvili, G., et al. 2019; Rosati, M., et al. 2021) and elsewhere in Europe as well (Beenken, L., et al. 2020; Mezzalama, M., et al. 2021; Voglmayr, H., et al. 2021).

First Report of Root Rot Caused by Macrophomina phaseolina in Peanut Shandong Province, China.

Peanut (Arachis hypogaea L.) is a widely grown oilseed crop of great agricultural importance worldwide. In July 2022, disease symptoms were observed on peanut roots in Laixi (36º85' N, 120º54' E), Shandong Province, China. About 25% of the plants showed various symptoms, including stem and root rot and blackening, microsclerotia on the stem, yellowing and wilting of leaves, and even death. Twenty diseased plants were collected to confirm the pathogen. Symptomatic roots were cut into small pieces, disinfested with 75% ethanol for 1 min and 0.5% NaClO for 2 min, rinsed three times with sterile water, dried on sterile filter paper, and then spread on potato dextrose agar (PDA) supplemented with 100 μg/mL chloramphenicol and incubated at 25°C in the dark. At the beginning of growth, the fungus formed sparse, white mycelia, which white, then darkened with age and microsclerotia were formed in the medium after 5 days. The mycelium aggregated into black, round to oblong or irregularly shaped microsclerotia 84 to 163 μm long and 54 to 125 μm wide (n=40). These morphological characteristics were consistent with the description of Macrophomina phaseolina (Holliday and Punithalingam, 1970). Molecular identification was performed by sequencing the internal transcribed spacer (ITS) region with ITS1 and ITS4 and translation elongation factor 1-alpha (TEF) with EF1-728F/EF1-986R (Glass and Donaldson 1995) of a representative isolate SXY183. ITS (OR056369) and TEF (OR098356) of SXY183 showed 100% and 97.74% similarity with M. phaseolina (KF951622, KF951997), respectively. Phylogenetic analysis was performed using Neighbor-Joining (NJ) analysis based on the gene sequences of ITS and TEF. The fungus was identified as M. phaseolina based on molecular analysis and morphological characteristics. The pathogenicity of a representative isolate (SXY183) was tested on peanuts under greenhouse conditions. Two-week-old peanut (Huayu No. 9115) seedlings were inoculated with a mycelial plug (8 mm diameter) at the root base of each plant and cultured in a greenhouse (30°C during the day and 25°C at night, a 12-h photoperiod, and 80% RH). Ten plants were inoculated with a plug of non-colonized PDA as a control. Brown lesions were observed on the stem and root of all inoculated seedlings 7 days after inoculation, but not on the control plants. The experiment was repeated three times. M. phaseolina was re-isolated from the symptomatic root and confirmed based on morphological characteristics and DNA sequence analysis of ITS and TEF. M. phaseolina is a soil-borne fungus that is distributed worldwide and has a broad host range. Disease agent has previously been reported on several host plants such as adzuki bean, faba bean, watermelon, Plukenetia volubilis, Atractylodes lancea and Curcuma longa in China (Cai et al., 2020; Sun et al. 2016; Sun et al., 2019; Sun et al., 2020; Wang et al., 2020; Wu et al., 2022). However, this is the first report in which M. phaseolina was found to cause peanut root rot in Shandong Province, China. Our report will provide important information for studying the epidemiology and management of this disease.

First report of Trunk Canker Caused by Botryosphaeria dothidea on Cinnamomum camphora in China.

Cinnamomum camphora, an essential woody plant in China, experienced a severe outbreak of trunk canker affecting an area of 540,000 square meters in the Xiaoshan District of Zhejiang province during the early summer of 2022. The observed symptoms included stem canker, dieback, twig blight, and extensive vascular discoloration, with an incidence rate ranging from 45% to 70%. To investigate the etiology of the disease, symptomatic plant samples were subjected to rigorous surface sterilization involving washing with running tap water, followed by surface sterilization using 75% ethanol. The samples were then rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA) medium, and incubated at a temperature of 28°C. The isolated fungi exhibited characteristics consistent with the species Botryosphaeria dothidea. Fungal isolates displayed abundant white aerial mycelium, which darkened to grey after five to seven days, eventually giving rise to black pycnidia. Single hyphal tip cultures of putative two isolates were stored at the Agricultural Experiment Station of Zhejiang University. Conidia formed on pycnidia were one-celled, hyaline, aseptate, and fusiform, with dimensions of 18.34-27.12 μm x 3.77-6.84 μm (average 22.90 μm x 5.20 μm) (one hundred conidia were measured). To determine the fungal species, genomic DNA was extracted from individual isolates ZJUP0868, and subjected to DNA sequence analysis of four gene regions: the internal transcribed spacer (ITS) using the primer-pair ITS4 and ITS1 (White et al., 1990), the large subunit (LSU) gene with LR0R and LR5 (Rehner and Samuels, 1995), the small subunit (SSU) gene with SR1R and SR7 (Zoller et al., 1999), and the translation elongation factor 1-alpha (tef1) with EF1-983F and EF1-2218R (Rehner and Buckley, 2005). Furthermore, DNA sequence analysis of above four genes was performed. BLAST analysis indicated the highest nucleotide sequence identity with Botryosphaeria dothidea CBS 115476 reference sequence (ITS: 99.59%, KF766151; LSU: 99.88%, DQ377852; SSU: 100%, NG_062738; tef1: 98.93%, AY236898). Representative sequences of isolate ZJUP0868 from these regions were deposited in GenBank (ITS: Accession No. OR192838; LSU: Accession No:OR248147; SSU: Accession No:OR248174; tef1: Accession No. OR262053). A phylogenomic analysis was conducted to determine the phylogenetic position of Botryosphaeria dothidea in the family Botryosphaeriaceae. The combined morphological and molecular findings confirmed the identification of the pathogen as Botryosphaeria dothidea. Pathogenicity tests were conducted by stem inoculation of two-year-old Cinnamomum camphora baranches. Mycelial plugs (2-3 mm in diameter) from actively growing colonies of B. dothidea (PDA) were applied to bark of similar size on the middle point of the stems. Inoculated barks were wrapped with Parafilm, while control branches received sterile PDA plugs. Inoculated and control branches (7 each) were maintained in a greenhouse at 28°C. After two weeks, all inoculated plants exhibited dark vascular stem tissue, whereas the control plants remained healthy. B. dothidea was re-isolated from symptomatic tissues, thus fulfilling Koch's postulates. No symptoms were observed in the control branched, and B. dothidea was not re-isolated from their tissues. B. dothidea , the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales), is commonly associated with cankers and dieback in woody plants. Previous reports have identified Botryosphaeria dothidea as a pathogen causing stem dieback and trunk canker on Glycine max (Chen et al., 2020), Camellia oleifera (Hao et al., 2022), and Gleditsia sinensis (Huang et al., 2020). Additionally, B. dothidea has been reported to cause leave wilt on various plant species in China, including Daimyo oak (Liu et al., 2023) and Cornus officinalis (Zhang et al., 2022). To the best of our knowledge, this is the first report of B. dothidea on C. camphora in China. This findings contribute to a better understanding of the pathogens affecting Cinnamomum camphora in the China.

First report of Bot Canker (Diplodia corticola) in Pin Oak (Quercus palustris) in Tennessee.

Diplodia corticola is a fungal pathogen contributing to oak (Quercus spp.) decline in the Mediterranean and US (Félix et al., 2017; Ferreira et al., 2021). In 2021, this pathogen was detected in Tennessee (TN) causing branch dieback in Q. alba (Onufrak et al., 2022). In September 2021, a matured pin oak (Q. palustris) with wilted leaves and elongated branch cankers was observed in the State Botanical Garden of Tennessee-Knoxville (TN, US). Small sections of the phloem were sampled from canker margins of a symptomatic branch using a sterile scalpel, surface sterilized, and plated onto potato dextrose agar amended with antibiotics (PDA++) (Gazis et al. 2018). Three days later, a fungal isolate resembling D. corticola was cultured on ½ PDA. Diplodia corticola is characterized on half-strength PDA by fast growth, irregular margins, and dense white mycelium that turns dark, grayish as the mycelium matures (Úrbez-Torres et al., 2010; Alves et al., 2004). Total genomic DNA was extracted from this isolate following Gazis et al. (2018), and the internal transcribed spacer (ITS), large ribosomal subunit (LSU), and transcription elongation factor 1-α (ef1-α) were amplified (Ferreira et al. 2021). Resulting PCR products were sequenced and assembled into consensus sequences using Unipro UGENE v. 44.0 (Okonechnikov et al., 2012). Each consensus sequence identity was determined using BLAST on the NCBI nucleotide database, restricted to type material. The ITS (accession OQ189888), ef-1α (accession OQ201608), and LSU (accession OQ189887) sequences had a 99.6% (accession KF766156.1), 98.6% (accession XM_020275852.1), and 100% (accession KF766323.1) identity match with D. corticola type culture CBS112549, respectively. To complete Koch's postulates and assess potential pathogenicity on economically and ecologically relevant oaks, 10 pin (Q. palustris; caliper 15.6 ± 2.0 mm), 10 overcup (Q. lyrata; caliper 15.1 ± 2.4 mm), and 10 sawtooth (Q. acutissima; 16.1 ± 2.1 mm) oaks were acclimated in the greenhouse for 1 week prior to the experiment. Five trees of each species were then randomly inoculated at 30 cm above the soil line with a 3 mm diameter plug of D. corticola (grown for 10 days on PDA; Sitz et al. 2017). To serve as a control, the remaining 5 trees for each species received a 3 mm diameter PDA plug. Fifteen days post-inoculation, seepage was observed in D. corticola-inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks. No seepage from wound sites was noted in control trees. Cankers were exposed, photographed, and then measured using ImageJ (Rasband, 2012). Using a sterile scalpel, four wood chips were excised from canker margins and plated onto PDA++. We recovered D. corticola from symptomatic inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks and confirmed species identity by extracting DNA and amplifying the ITS, ef-1α, and LSU regions as described above (Gazis et al., 2018; Ferreira et al., 2021). The resulting consensus sequences matched the D. corticola type culture (CBS112549) ITS (99.0%-99.8% identity), ef-1α (91.0%-99.1% identity), and LSU (96.9%-100% identity) barcoding regions. Cankers were significantly larger in D. corticola-inoculated pin (4.7 ± 1.5 cm2; P = 0.003), overcup (6.8 ± 2.9 cm2; P = 0.009), and sawtooth (5.1 ± 1.3 cm2; P = 0.001) oaks in comparison to the control trees from these groups. Based on current reports, this is the first record of D. corticola causing dieback in pin oak (Q. palustris) in TN.

First report of Sclerotinia sclerotiorum causing Sclerotinia wilt of hop (Humulus lupulus) in Poland.

The common hop (Humulus lupulus L., Cannabaceae) is a perennial plant cultivated in the temperate climate zone and used in the brewing and pharmaceutical industry. In June 2021, symptoms of wilting and subsequent drying of shoots were observed on hop plants (cv. Lubelski) in Lubelskie Province, in Poland (50°55'30.5"N, 22°10'35.4"E). Wilted shoots showed no symptoms of chlorosis. Usually both healthy and wilted shoots were present on the same plant. Approx. 20% of the plants in the 2 hectare hop garden showed these symptoms. An inspection of the underground parts of the infected shoots revealed a brown necrosis of the tissues partly covered by white mycelium with black sclerotia. These symptoms were characteristic for Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006), but this pathogen has never been detected in hop gardens in Poland before. In order to confirm the preliminary diagnosis, shoots from five different plants with symptoms of necrosis were collected, disinfected with 1% sodium hypochlorite for 1 min, rinsed with sterile water and dried. Twelve fragments from partially necrotized tissue were placed on potato dextrose agar (PDA) amended with chlortetracycline hydrochloride. After three days of incubation at room temperature and daylight, 75% of the explants had white or light grey mycelia present. Pure cultures were obtained by transplanting mycelium to fresh PDA. Within 4 days, the mycelium has reached the diameter of Petri dishes (90 mm). On PDA at room temperature and daylight, black spherical or cylindrical sclerotia formed after 6-14 days. Sclerotial size was 1.0 to 10.0 x 1.0 to 5.0 mm (average 2.8 x 2.0 mm; n = 57 from 3 plates). Five isolates were subjected to DNA extraction. Then ITS region was amplified and sequenced using primers ITS1/ITS4 (White et al. 1990). The sequences obtained from the five fungal isolates were identical with each other and BLAST analysis revealed also 100% identity with the GenBank records of S. sclerotiorum (e.g. KT224645, Baturo-Ciesniewska et al. 2017). One representative sequence of isolate Ss5HL was deposited to GenBank (OQ998981). Pathogenicity of the isolate Ss5HL was confirmed in inoculation tests on approx. 30 cm high, young shoots of hop plants (cv. Lubelski) grown from rootstock for 4 weeks in the greenhouse. Five plants were inoculated by placing mycelial discs from a 7-day-old culture on shoots and covering them with wet sterilized cotton pads and aluminum foil. Two control plants were treated in the same way but instead of mycelial discs pure agar discs were placed on the shoots. Then plants were kept in a climate chamber (80% relative humidity, 15-h light at 22°C, 9-h dark at 18°C). All fungal inoculated shoots wilted within 3-4 days, then necrosis developed and spread from the place of inoculation up the shoot. No tissue necrosis was observed below the place of inoculation. The control plants remained asymptomatic. The inoculation test was carried out twice with the same result. Fungal cultures reisolated from the inoculated shoots were morphologically identical with the culture used as inoculum. Isolate Ss5HL was deposited in the collection of the Westerdijk Fungal Biodiversity Institute (CBS 150077).To our knowledge, this is the first case of Sclerotinia wilt of hops in Poland and also in Europe, with the exception of one, nearly 100 years old report from the United Kingdom (Salmon and Ware 1936). S. sclerotiorum rarely occurs on hop but it can cause severe yield reduction if pathogen accumulates in the soil.

Fruit rot causing by Athelia rolfsii (Sclerotium rolfsii) on jackfruit (Artocarpus heterophyllus) in China.

Artocarpus heterophyllus, known as jackfruit, was a tropical fruit and cultivated extensively as nutritional and medicinal properties in southern China in recent year. During July 2022, fruit rot was observed on the fruits at the bottom of jackfruit trees in an orchard in Zhanjiang, Guangdong (N21°9' 27" E110°17' 54") 3-4 days after typhoon. The incidence rate of fruit was about 0.3%. The initial symptom was white mycelia appearing on the surface of fruits. Mycelia with rhizomorphs spread rapidly over the fruits, formed white, often fan-shaped mats with the rapeseed size sclerotia. The infected fruits were water-soaked, quickly became rotten, and fell off. Sclerotia from disease fruits were incubated on PDA with 50 mg/L ampicillin at 25-28℃ in the dark for 2 days. Hyphae tips were transferred to get the purified isolates. Colonies with a radial growth rate of 23.2 mm/day had abundant aerial mycelia and profuse sclerotia on PDA. Hyphae of the isolates were transparent, branched, with clamp connections at septa, usually 2.9-8.3 µm (Ave. 5.8 µm) (n>30) wide. Aerial mycelia were whitish-cottony, with many narrow rhizomorphs. Spherical sclerotia developed at about 10 days after incubation, and gradually changed from white to tan-to-dark brown, and mature sclerotia were about 1.7 mm in size. The morphological characteristics was similar to those of Sclerotium rolfsii (teleomorph: Athelia rolfsii). To accurately identify the fungus, the internal transcribed spacer gene (ITS) and large subunit rRNA gene (LSU) of isolate CASS-BLM-1 were PCR amplified with primer pairs ITS1/ITS4 (White et al 1990) and V9G/LR5 (Klaubauf et al 2014). The amplicons were sequenced and deposited in GenBank with accession number OP535473 (ITS) and OP535474 (LSU). BLASTn results showed that the nucleotide sequences of ITS and LSU had high identity with corresponding sequences of A. rolfsii isolates CBS 191.62 (ITS: MH858139, 472/474(99.58%); LSU: MH869724, 882/885(99.66%)) (Vu et al 2019). Phylogenetic analysis based on ITS sequence data was obtained according to maximum likelihood method using MEGA analysis software, CASS-BLM1 was grouped in A. rolfsii clade with 100% bootstrap support value. Based on morphology and DNA sequences, the fungus was identified as A. rolfsii (anamorph: S. rolfsii). To fulfil Koch's postulates, healthy fruits on the tree and detached fruits were inoculated with 7-day-old sclerotia of isolate CASS-BLM1. Five unwound sites and five wound sites with a sterile needle were tested on each fruit and a sclerotium was put at each site. Fruits not inoculated with sclerotia were used as control the test was repeated three times. All fruit were enclosed in transparent plastic bags with sterile absorbent cotton moistened with sterile distilled water. The indoor and outdoor temperatures ranged from 25 to 30 ℃. Three days later, white mycelia were observed on all inoculation sites, and 5 days later, the inoculated fruits began to rot, while control fruits remained healthy. The same fungus with identical morphology and DNA sequences was re-isolated from the inoculated sites. Previously, A. rolfsii was reported to cause fruit rot disease on jackfruit in Bangladesh (Elahi et al 2021), this is the first report of A. rolfsii causing fruit rot on jackfruit in China. A. rolfsii is suitable for high temperature and humidity environment (Punja 1985), this report will help farmers to diagnose this disease, especially to strengthen the disease prevention during the typhoon season.

First Report of Muskmelon Fruit Rot Caused by Fusarium sulawesiense in China.

Muskmelon(Cucumis meloL.) is a widely cultivated and economically important fruit crop worldwide. In June 2022, fruitrot symptoms were observed on ripening muskmelons (cv. Boyang) in Shouguang City (36.81°N 118.90°E) of China. To determine the causal agent, we surveyed 200 muskmelon plants in about 1000 m2 of planting area and collected diseased muskmelons. Approximately 20% of muskmelon fruits had symptoms, and yield loss averaged 20%. Water-soaked lesions were observed on the surface and the fruit rotted from inside. Lesions were covered with white mycelium. Rotted fruit were surface-disinfested with 1% NaOCl for 1 min, 75% ethanol for 30 s, and washed three times with sterile water. Pieces (1 cm3) were cut from the disinfested fruit, placed on potato dextrose agar (PDA), and incubated at 25°C for 1 week. Ten isolates with similar morphology were obtained and isolates SG66 and SG68 were selected for further characterization. Colonies maintained on PDA in the dark had an average radial growth rate of 10-12 mm/d at 25°C. Surface was white, velvety to felty mycelium. Reverse was white to pale wheat. Diffusible pigments were absent. On carnation leaf agar, sporodochia appeared as slimy dots, macroconidia were 3- to 5-septate, 20-35 × 3-5 μm, falcate, with a pronounced dorsiventral curvature, with blunt to papillate apical cell, and barely to distinctly notched basal cell. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with descriptions of Fusarium sp. DNA was extracted from isolates SG66 and SG68 using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using generic primers, the products sequenced, and sequences deposited in GenBank (ITS: OP251362, OP251363; CAM: OP266024, OP266025; RPB2: OP266028, OP266029; TEF1: OP266026, OP266027). Isolates SG66 and SG68 clustered with Fusarium sulawesiense (85% bootstrap) (Maryani et al. 2019). The Fusarioid-ID database pairwise alignment of ITS (526 bp), CAM (534 bp), RPB2 (861 bp), and TEF1 (636 bp) sequences from isolate SG66 showed 99.6% (98.9% coverage), 100% (100% coverage), 100% (100% coverage) and 100% (98.4% coverage) similarity with the corresponding sequences (GQ505730, LS479422, LS479855 and GQ505641), respectively, of the reference strains of F. sulawesiense (InaCC F940 and NRRL 34059). To perform a pathogenicity test, 10 μl of conidial suspensions (1 × 106 conidia/ml) were injected into ten muskmelon fruit using a syringe, and ten control fruit were inoculated with 10 μl of sterile distilled water. The test was repeated three times. After 7 days at 25°C, the pulp of all inoculated muskmelons began to rot, and the lesion expanded from the inside to the fruit surface at the injection site and became covered with white mycelia. No symptoms developed on the control fruit. The fungus was successfully re-isolated from infected tissues and confirmed as F. sulawesiense by morphological and phylogenetic analyses. F. sulawesiense has previously been reported onyellow melon (Canary) in Brazil (Lima et al. 2021) and on a range of hosts, including Luffa aegyptiaca, in China (Wang et al. 2019). To our knowledge, this is the first report of muskmelon fruit rot caused by F. sulawesiense in China.

First Report of Fusarium oxysporum Causing Leaf Wilt on Dinteranthus vanzylii (Green Stone Plant) in China.

Dinteranthus vanzylii is a low-growing species in the family Aizoaceae, native to southern Africa, with a pair of thick grey leaves covered with dark red spots and stripes. This stone-like succulent grows near the ground, which may protect it from water evaporation and herbivores. Dinteranthus vanzylii has become popular in China due to its attractive appearance and easy indoor cultivation. In September 2021, 7% of D. vanzylii (approximately 140 pots) showed leaf wilt symptoms in a commercial greenhouse located in Ningde (119°35'39.696″E, 27°23'30.556″N), Fujian Province, China. The diseased plants were shrivelling and eventually underwent necrosis. Their leaf tissues were rotting and carpeted with white mycelium. The leaf tissues of 10 symptomatic plants were cut into 0.5 cm2 pieces, surface-sterilized and placed on PDA medium. According to the colony morphology after 7 days of culture, 20 fungal isolates with abundant whitish aerial mycelium were divided into two types: 8 isolates produced lilac pigment whereas 12 did not. Both produced unicellular ovoid microconidia, sickled-shaped macroconidia with 3 - 4 septa and single or paired smooth, thick-walled chlamydospores on carnation leaf agar (CLA). Molecular identification based on DNA sequences from EF1-α (O'Donnell et al. 1998), RPB1 and RPB2 (O'Donnell et al. 2010) revealed 100% identity among isolates within each group; however, there were several base differences between two types. Sequences of representative isolates KMDV1 and KMDV2 were deposited in GenBank (acc. nos.: OP910243, OP910244, OR030448, OR030449, OR030450 and OR030451), which showed 99.10% - 99.74% identity with different F. oxysporum strains (GenBank acc. nos.: KU738441, LN828039, MN457050, MN457049, ON316742 and ON316741). Phylogenetic tree inferred from the concatenated EF1-α, RPB1 and RPB2 revealed that these isolates clustered with F. oxysporum. Thus, these isolates were identified as F. oxysporum. Using a root-drenching method, 10 one-year-old healthy D. vanzylii were inoculated in conidial suspensions (1*106 conidia/mL) of isolates KMDV1 and KMDV2 for 60 min, respectively. They were transplanted into pots with sterilized soil and incubated in a plant-growth chamber at 25°C and 60% relative humidity. Control plants were treated with sterilized water. The pathogenicity test was repeated three times. All plants inoculated with each isolate developed leaf wilt symptoms after 15 days and were dead after 20 - 30 days. However, no symptoms were observed in the control plants. Fusarium oxysporum was reisolated and confirmed based on morphology and EF1-α sequence analysis. No pathogens were isolated from the control plants. This is the first report of F. oxysporum causing leaf wilt disease on D. vanzylii in China. To date, several diseases have been reported on members of the Aizoaceae. For instance, collar and stem rot on Lampranthus sp. caused by Pythium aphanidermatum (Garibaldi et al. 2009), wilt on Lampranthus sp. and Tetragonia tetragonioides caused by Verticillium dahliae (Garibaldi et al. 2010; Garibaldi et al. 2013), and leaf spot on Sesuvium portulacastrum caused by Gibbago trianthemae (Chen et al., 2022). Our research could provide insight into fungal diseases on members of the Aizoaceae and contribute to their cultivation and management.

First Report of White Mold Caused by Sclerotinia sclerotiorum on Brussels Sprouts in Mexico.

The Brussels sprout (Brassica oleracea var. gemmifera) is a cruciferous vegetable with high health-promoting value and Mexico is one of the most valuable exporters worldwide (Data Mexico 2023). From September to November 2021, white mold symptoms (Rimmer et al. 2007) were observed in Brussels sprouts (cv. Confidant) fields in Tonatico, Estado de México, Mexico. Irregular, necrotic lesions were observed on leaves, whereas abundant white mycelium, and later black sclerotia were produced outside and inside of stems. Disease incidence ranged from 20 to 40% in five fields. For fungal isolation, symptomatic stem pieces were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed in sterilized distilled water twice, placed on PDA medium, and incubated at 25°C in darkness for 3 days. Sclerotinia-like colonies were consistently obtained and six isolates were purified by the hyphal-tip method. Fungal colonies were white and fluffy. Irregular, black, and small (3 to 6 mm diameter) sclerotia were produced at the edge of colonies after 5 days of incubation. The morphological characters were consistent with those of Sclerotinia sclerotiorum (Saharan and Mehta 2008). Two representative isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero under the accession numbers CSAEG50 and CSAEG51. For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region was amplified by PCR and sequenced using the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OQ878510 and OQ878511). BLASTn searches in GenBank showed 100% identity with the available sequences of Sclerotinia sclerotiorum (accession nos. OQ891471, OQ891472, HQ833448, and MT177216). A phylogenetic analysis using the Maximum Likelihood method placed isolates CSAEG50 and CSAEG51 in the same clade as S. sclerotiorum. Pathogenicity tests were performed by inoculating 10 healthy Brussels sprout seedlings (cv. Confidant) grown in pots. A mycelial plug was directly placed on the stem of each plant. Five uninoculated Brussels sprout seedlings were used as control. All plants were placed in a moist chamber at 25°C with a 12-h photoperiod for 2 days. White mold symptoms appeared on inoculated plants after 3 days, whereas control plants remained symptomless. The fungi were reisolated from the infected plants and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch's postulates. Pathogenicity test was performed twice with similar results. Sclerotinia sclerotiorum has been previously reported to infect Brussels sprouts in the USA (Campbell 1947). To our knowledge, this is the first report of Sclerotinia sclerotiorum causing white mold of Brussels sprouts in Mexico. The disease is widely distributed in Brussels sprouts fields in the central region of Mexico, therefore additional studies are needed to develop effective disease-management strategies.

First Report of Colletotrichum gloeosporioides and Colletotrichum siamense, Causing Anthracnose Disease on coffee trees, in Saudi Arabia.

Coffee (Coffea arabica L.) is a promising agricultural commodity in many countries including Saudi Arabia, but crop production is often constrained by diseases. In December 2021, coffee trees had symptoms of anthracnose disease (CAD) were observed in Jazan Province, Saudi Arabia (17°19'00.8"N 43°11'26.8"E), and the incidence was 55%. Affected trees showed dieback and leaves necrosis. On green and ripening berries, slightly sunken and dark brown lesions were occurred; the berries finally become mummified (Fig. S1). For pathogen isolation, symptomatic tissues (4×4mm) of 30 diseased branches and berries samples were surface-sterilized in 1% sodium hypochlorite for 2 min, followed by 70% ethanol for 20 s, rinsed in sterile distilled water and placed on potato dextrose agar (PDA). Cultures were incubated at 26℃ for 8 days in the dark. Eighteen isolates were recovered, and 2 representative single spore isolates (KSU-CgM17, KSU-CsM42) were used for further study. PDA culture of KSU-CgM17 had aerial white mycelium at first and later became gray to grayish black; light salmon to orange conidial masses were observed on the mycelium plate surface as the cultures aged (Fig. S2). Colony produced by KSU-CsM42 was off-white to gray with cottony mycelia and grayish-white on the undersides of the culture after 10 days at 28° (Fig. S2). Conidial shape of these two isolates were both aseptate, cylindrical to nearly straight, hyaline, rounded at both ends. Conidia (n = 50) measurements were 16 to 18.0 µm long × 4.8 to 6.4 µm wide for KSU-CgM17 and 12.6 to 17.5 µm long × 3.2 to 4.5 μm wide for KSU-CsM42. The microscopic and culture features fitted those for Colletotrichum gloeosporioides species complex (Weir et al. 2012). To further identify these isolates, four genomic DNA loci including the partial ITS rDNA region, and CAL, TUB2, and GAPDH genes were amplified and sequenced (Hu et al., 2015). All sequences were deposited into GenBank under accession numbers: OQ791412 & OQ791413 (ITS), OQ786847 & OQ786851 (CAL), OQ786849 & OQ786850 (TUB2), and OQ786848 & OQ786852 (GAPDH) for KSU-CgM17and KSU-CsM42, respectively (Tables S1& S2). A BLAST search of GenBank showed that these pathogens were identified as C. gloeosporioides (KSU-CgM17) and C. siamense (KSU-CsM42). The pathogenicity was tested on detached coffee leaves or green and red berries (Coa et al., 2019). For inoculation, healthy leaves and berries were wounded with a sterilized needle, placed inside petri dishes containing moist filter paper, and then inoculated with a 10-µl droplet of conidial suspension (106 spores/ ml). Sterile distilled water was used as a negative control. Six replicates were tested per isolate and the experiment was repeated once. The inoculated materials were incubated at 25°C and 100% relative humidity for 8 days. Necrotic lesions developed on 100% of the inoculated coffee materials 6 days later, whereas the negative controls were asymptomatic (Fig. S2). Koch's postulates were fulfilled when typical colonies of these species were successfully re-isolated from the from symptomatic tissues. These pathogens were reported previously to affect coffee in Vietnam (Nguyen et al., 2010), China (Cao et al., 2019), and Puerto Rico (Serrato-Diaz et al., 2020). To our knowledge, this is the first record of C. gloeosporioides and C. siamense causing CAD in Saudi Arabia. Further studies on the epidemiology of CAD on arabica coffee plantations as well as effective strategies for managing this disease are needed.

Morphocultural and molecular characterization of Athelia rolfsii associated with foot rot disease of Gomphrena globosa – A new record from India

Globe amaranth is an important floriculture plant grown for the cut flowers. The outbreak of southern blight disease was observed during 2019 in parts of the southern Karnataka. The disease incidence ranged from 9.54 to 21.13%. Disease symptoms include water-soaked lesions on the lower leaves, followed by quick wilting of the whole plant. The fungal pathogen was isolated on potato dextrose agar medium which produced white cottony mycelium with numerous reddish-brown sclerotia developed after 12 days of inoculation. A total of 12 fungal isolates were obtained from infected samples and their identity was established based on micro-morphological studies. Further, three isolates were used for molecular identification by sequencing the ITS-rDNA and tef-1α gene showed 100% sequence similarity with reference sequences. The associated fungal pathogen was identified as Athelia rolfsii. Further, pathogenicity tests were conducted on healthy plants in-vitro and in green house conditions. Typical foot rot disease symptoms were observed after 8 days of post-inoculation. The pathogen was re-isolated and its identity was confirmed. There are no reports of A. rolfsii on globe amaranth. To the best of our knowledge, this is the first report of A. rolfsii associated with southern blight disease of globe amaranth in India.

First Report of Cucurbita Blossom Blight and Fruit Rot Caused by Choanephora cucurbitarum in Mexico.

In December 2022, blossom blight, abortion, and soft rot of fruits were observed on Cucurbita pepo L. var. Zucchini in Mexico under greenhouse conditions (temperatures of 10 to 32°C and relative humidity up to 90%). The disease incidence in about 50 plants analyzed was around 70% with a severity of nearly 90%. Mycelial growth on flower petals and fruit rot with brown sporangiophores was observed. Ten disinfested fruit tissues in 1% NaClO for 5 min and then rinsed twice in distilled water from the lesion edges were placed on potato dextrose agar culture medium (PDA) supplemented with acid lactic and then, the morphological characterization was carried out in V8 agar medium. After 48 h of growth at 27°C, the colonies were pale yellow with diffuse cottony mycelia that were non-septate and hyaline and produced both sporangiophores bearing sporangiola and sporangia. The sporangiola were brown, ranged from ellipsoid to ovoid, and had longitudinal striations that measured 22.7 to 40.5 (29.8) μm x 16.08 to 21.9 (14.5) μm long and wide, respectively (n=100). The sporangia were subglobose, had a diameter of 127.2 to 281.09 (201.7) μm (n=50), and contained ovoid sporangiospores that measured 26.5 to 63.1 (46.7) μm x 20.07 to 34.7 (26.3) μm long and wide, respectively (n=100) which had hyaline appendages at the ends. Based on these characteristics, the fungus was identified as Choanephora cucurbitarum (Ji-Hyun et al. 2016). For molecular identification, DNA fragments for the internal transcribed spacer (ITS) and the large subunit rRNA 28S (LSU) regions for two representative strains (CCCFMx01 and CCCFMx02) were amplified and sequenced with the primer pairs ITS1-ITS4 and NL1-LR3 (White et al. 1990; Vilgalys and Hester 1990). The ITS and LSU sequences were deposited in GenBank database (Accession numbers OQ269823-24 and OQ269827-28, respectively) for both strains. The Blast alignment showed from 99.84 to 100% identity with Choanephora cucurbitarum strains JPC1 (MH041502, MH041504), CCUB1293 (MN897836), PLR2 (OL790293), and CBS 178.76 (JN206235, MT523842). To confirm the specie identification, the evolutionary analyses were conducted from the concatenated sequences of the ITS and LSU of C. cucurbitarum and other mucoralean species with the Maximum Likelihood method and Tamura-Nei model included in the software MEGA11. The pathogenicity test was demonstrated using five surface-sterilized zucchini fruits inoculated with a sporangiospores suspension containing a concentration of 1 x 105 esp/mL on two sites per fruit (20 µL each) that previously were wounded with a sterile needle. For fruit control, 20 µL of sterile water was used. Three days after inoculation under humidity conditions at 27°C, white mycelia and sporangiola growth with a soaked lesion were observed. That fruit damage was not observed on the control fruits. C.cucurbitarum was reisolated from lesions on PDA and V8 medium which was confirmed by morphological characterization fulfilling Koch's postulates. Blossom blight, abortion, and soft rot of fruits caused by C. cucurbitarum were observed on Cucurbita pepo and C. moschata in Slovenia and Sri Lanka (Žerjav and Schroers 2019; Emmanuel et al. 2021). This pathogen has the capability to infect a wide variety of plants worldwide (Kumar et al. 2022; Ryu et al. 2022). There are no reports of C. cucurbitarum causing agricultural losses in Mexico, and this is the first report causing the disease symptoms in Cucurbita pepo in this country; however, this fungus was found in the soil of papaya-producing areas and it is considered an important plant pathogenic fungus. Therefore, strategies for their control are highly recommended to avoid spreading the disease (Cruz-Lachica et al. 2018).

First Report of Alternaria alternata Causing Leaf Spot on Broccoli in Ecuador.

In Ecuador, broccoli (Brassica oleracea var. italica) production is located in the Andean region, specifically Cotopaxi-Ecuador (INEC, 2019). A leaf pathogen has been constantly observed in this area, showing brown circular necrosis surrounded by yellowish halo-like spots causing leaf death (Fig. 1a). This pathogen was believed to be Alternaria sp.; however, the species was not determined either using classical or molecular tools. In 2021, ten leaves showing similar symptoms were collected in Cotopaxi and sent for pathogen identification. Here, leaf explants (0.25 cm2) showing disease symptoms were surface sterilized with 2% sodium hypochlorite (NaClO) and 70% ethanol (C2H6O), rinsed with sterile water, and transferred to Potato Dextrose Agar (PDA) media. Petri dishes were incubated in darkness at 25°C for five days. The single hyphal tip method was used to purify the cultures on PDA. Fifteen pure isolates were obtained after incubating for 14 days. Isolates were incubated under blacklight for two days to induce fungal sporulation. All isolates presented early white cotton-like mycelium that later became dark green (Fig 1b). Under the microscope, we observed straight primary conidia in simple or branched chains. Also, the conidia were obclavate, long ellipsoids, moderate in size (19.5-43.9 μm in length, 7.1-17.2 μm in width), and septate with few longitudinal septa. Lastly, the conidium body can narrow itself into a secondary conidia (Fig 1c) (Woudenberg et al., 2013). According to colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al., 2013). Five isolates were randomly selected for DNA extraction and sequencing of ITS (internal transcribed spacer; Chou, H.H. and Wu, W.S. 2002), TEF (translation elongation factor; O'Donnell et al., 1998), and RPB2 (RNA polymerase II second largest subunit; Liu et al., 1999) gene regions. DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al., 2018). Consensus sequences were deposited in GenBank with the following accession numbers: ITS, ON982232; TEF, ON983964; RPB2, ON983963. A multilocus Bayesian inference phylogenetic tree was constructed in Beast software (version 1.8.4) using the concatenated sequences (Drummond et al., 2012; Maharachchikumbura et al., 2014). The isolates in our study clustered with isolates of Alternaria alternata, confirming their identity (Figure 2). For Koch's postulates, healthy broccoli plants were grown in sterile soil for six weeks. The fungal conidia were suspended in sterile distilled water (1×106 conidia/ml), and the leaves were inoculated by spraying the spore solution. The control treatment was sprayed with sterile distilled water alone. Plants were maintained at 28°C and had more than 85% relative humidity (Sigillo et al., 2020). Seven days after inoculation, plants showed chlorosis and necrosis. Ten days later, 100% of the treated leaves presented brown circular necrosis (Fig. 1d). Control plants showed no disease symptoms. Re-isolation of the pathogen from the diseased leaf tissue was performed as previously described. The isolates presented the exact morphology of pure cultures obtained from field-diseased leaves. The pathogenicity test was performed twice. To our knowledge, this is the first report on A. alternata being the causal agent of leaf spot on broccoli in Ecuador. Disease diagnosis contributes to providing strategies against this pathogen. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in broccoli.

First report of Fusarium keratoplasticum causing strawberry root rot in Sinaloa, Mexico.

Strawberry (Fragaria × ananassa) is a fruit of economic importance for Mexico, occupying the third place in world production, with an approximate production of 861, 337 t (SIAP, 2021). In January 2021, in Culiacan, Sinaloa, Mexico (24°46'46″N; 107°27'04″ W), wilting symptoms (stunted growth, leaf yellowing and wilting, necrosis in vascular bundles, root rot and wilting) were observed on commercial strawberry crops, with an incidence of 5 to 10 %. Tissue samples from symptomatic roots were cut and disinfected with alcohol, sodium hypochlorite and sterile water, to later be plated on potato dextrose agar (PDA). Fifteen monosporic isolates were obtained by single-spore culturing (Hansen and Smith, 1932). Typical Fusarium spp. colonies were obtained from all root samples. On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septate, and light-yellow pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidia were slightly curved, showing three marked septa, broad central cells, slightly tapered apices, foot-shaped basal cells and measured 59.6 - 73.4 (x̄ = 68.7) x 10.4 - 14.9 μm (x̄ = 13.6) (n = 40). The microconidia (n = 40) were thin-walled, hyaline, ovoid unicellular that measured 19.7 - 32.2 (x̄ = 26.6) x 8.8 - 11.8 μm (x̄ = 10.2). The translation elongation factor 1 alpha (EF1-α) gene (O'Donnell et al. 1998) was amplified by polymerase chain reaction and sequenced. Maximum likelihood analysis was carried out using the EF1-α sequence from the isolate FKTFRESCULSIN (GenBank accession no. OK491929) and other Neocosmospora and Fusarium species. Phylogenetic analysis revealed the isolate was Fusarium keratoplasticum (currently named Neocosmospora keratoplastica) belonging to the Fusarium solani species complex (FSSC). Pathogenicity tests were performed on strawberry plants (cultivar Camarosa) grown on autoclaved sandy loam soil mix. Twenty plants were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) in an isotonic saline solution of FKTFRESCULSIN grown on PDA. Ten uninoculated plants served as controls. Plants were maintained for 60 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Root and stem rot similar to that observed on the infected plants in the field was observed. No symptoms were observed on uninoculated control plants after 60 days. The pathogen was reisolated from necrotic tissue from all inoculated plants and identified as F. keratoplasticum by sequencing the partial EF1-α gene and based on its morphological characteristics, thus fulfilling Koch's postulates. To our knowledge, this is the first report of root rot and wilt of strawberry caused by F. keratoplasticum in Mexico; it also contributes knowledge to the scientific community, since there is little information about this pathogen causing damage to plants in the world. Strawberry is an important crop in Mexico, and the occurrence of this disease could threaten strawberry production.

First Report of Fusarium acuminatum Causing Leaf Spot on Schisandra chinensis in China.

Schisandra chinensis (Turcz.) Baill. is a popular and widely cultivated medicinal herb in China, which has rich nutritional value and medicinal effect. In August 2022, leaves with oval and irregularly circular light brown spots from 2 to 10 mm wide with white centers were found on Schisandra chinensis growing in Fusong district (127°28'E, 42°33'N) of Jilin, China. The symptoms were observed in 20% of the plants of a 2 ha-1 field of Schisandra chinensis. About 50% of the leaf areas were affected. As the disease developed, the lesions grew larger and developed necrotic centers. Leaves with light brown spot symptoms from five plants were collected from the field. Five leaf pieces (3 to 5 mm2) were excised from lesion margins, surface sterilized based on Ju et al. (Ju et al. 2021), and incubated on potato dextrose agar (PDA) at 25°C. Six single spores were isolated from five independently infected isolates for pure culture using the single spore isolation technique (Zhang. 2003). Representative single spore isolate (ZWWZH) was selected from pure cultures for further culture. After 5 days, fluffy white aerial mycelium with pink pigmentation on the underside of the colony were observed on PDA. Mycelia became pinkish-brown as the culture aged. Microscopic observations showed the presence of elongated or pointed, and thick-walled macroconidia (n = 50), predominantly three septate, 3.40 to 7.50 × 40.34 to 61.29 μm were observed. Chlamydospores formed in chains within or on top of the mycelium. The primers ITS1/ITS4 (White et al. 1990) and Bt-2a/Bt-2b (Robideau et al., 2011) were used to amplify the internal transcribed spacer (ITS) rDNA and β-tubulin (TUB2) region, respectively. The obtained sequences were submitted to GenBank under accession numbers for OQ629789 (ITS) and OQ803521(TUB2). BLASTn analysis of both ITS sequence and TUB2 sequence, revealed 100% and 99.92% sequence identity with F. acuminatum MT566456, MT560377 and KJ396328, respectively. The pathogen was identified as F. acuminatum based on morphological and molecular data. Pathogenicity tests were carried out in the greenhouse. Select five healthy Schisandra chinensis seedlings, each with each healthy leaf surfaces inoculated a 1 × 106 spores/mL solution, 3 wells on one side, 10 µL per well. Sterile ddH2O was used in the control experiment. The inoculated seedlings were incubated at 25°C with a relative humidity of 65 to 70% in a greenhouse. Four days after inoculation, all inoculated leaves exhibited the same symptoms as observed in the field, while the controls showed no symptoms. The experiment was repeated three more times with similar results. The re-isolated fungi from the inoculated plants had the same morphology and DNA sequences as the original isolate (ZWWZH) obtained from the field samples, completing Koch's postulates. To our knowledge, this is the first report of F. acuminatum causing leaf spot on Schisandra chinensis in China. F. acuminatum has seriously affected the quality of Schisandra chinensis production. The identification of leaf spot caused by F. acuminatum will enable farmers to identify practices to minimize disease on this important crop.

First Report of Leaf blight Caused by Colletotrichum karstii on Banana Shrub (Michelia figo) in Sichuan, China.

Banana Shrub (Michelia figo (Lour.) Spreng.) is widely cultivated in most of southern China (Wu et al, 2008). It can be used to make essential oil and flower tea（Ma et al, 2012; Li et al, 2010）.The first symptoms were observed in Sept. 2020 at a grower's field in Banana shrub seedlings (0.6 ha), Ya'an city (29°30'N, 102°38'E), Hanyuan county. The symptoms re-occurred in May-June of 2021 and became prevalent from August to September. the incidence rate and the disease index were 40% and 22%, respectively. Initially, purplish-brown necrotic lesions appeared at the leaf tip with dark-brown edges. Progressively, necrosis spread, to the middle of the leaves, and the older area turned gray-white. Dark sunken lesions appeared in the necrotic areas and orange conidial masses were visible under humid conditions. Ten isolates were obtained on potato dextrose agar (PDA) from 10 leaf samples using previously described tissue isolation method (Fang et al. 1998). All the 10 isolates exhibited similar morphological characteristics. Grey to white aerial mycelium at the center and in dispersed tufts, with numerous dark conidiomata scattered over the surface, reverse was pale orange with numerous dark flecks corresponding to the ascomata, orange conidial masses were formed from mature conidiomata. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical, apex round, the contents appearing granular 14.8 to 17.2 × 4.2 to 6.4 μm (average: 16.26 × 4.84 μm, n=30) as Colletotrichum spp. (Damm et al. 2012). For molecular identification, DNA was extracted from a representative isolate HXcjA using a plant genomic DNA extraction kit (Solarbio, Beijing). and the partial sequences of internal transcribed spacer region (ITS, OQ641677), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, OL614009), actin (ACT, OL614007), beta-tubulin (TUB2, OL614011), histone3 (HIS3, OL614010), and calmodulin (CAL, OL614008) were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT-512F/ACT-783R, CAL 228F/CAL 737R (Carbone et al. 1999), TUB1F/Bt2bR, CYLH3F/CYLH3R (Crous et al. 2004), respectively. BLASTn analysis for ITS, GAPDH, CAL, ACT, TUB2 and HIS3 sequences showed ≥99.7% identity to C. Karstii, namely, NR_144790 (532/532 bp), MK963048 (252/252 bp), MK390726 (431/431 bp), MG602039 (761/763 bp), (KJ954424, 294/294 bp), (KJ813519, 389/389 bp), respectively. The fungus was identified as C. karstii based on morphology and a multigene phylogeny. The conidial suspension (1 × 107 conidia/mL) with 0.05% Tween 80 buffer was used for pathogenicity test, by spraying 2-year-old Banana Shrub plants. Ten plants were inoculated with spore suspensions (approximately 2ml per plant). An equal number of plants were sprayed with 0.05% Tween 80 buffer to serve as a control. Fifteen days later, the inoculated plants showed similar symptoms as the original diseased plants but the controls remained asymptomatic. C. karstii was re-isolated from the infected leaves and identified by morphology and a multigene phylogeny. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of Banana Shrub leaf blight caused by C. karstii in China. This disease reduces the ornamental and economic value of Banana Shrub, and this work will provide a basis for the prevention and treatment of the disease in the future.

First report of Fusarium verticillioides causing safflower root rot in Sinaloa, Mexico.

Safflower cultivation is of great socioeconomic importance worldwide. Production is intended for the extraction of oil from the seeds. In 2021 Mexico ranked fifth in world production with approximately 52,553.28 tons (SIAP, 2021). In April 2022, in the north-central zone of Sinaloa, Mexico, diseased plants were reported in fields planted with safflower. Symptoms included chlorotic plants, necrosis and rot in vascular bundles, dwarfed plants and reflexed plants bent towards the ground. The disease caused estimated losses of 15% of seed production, with respect to the production obtained from the previous year in the safflower fields surveyed. Twenty-five plants with symptoms were sampled to isolate the pathogen. Plants were cut at the base of the stem near the roots and roots cut into 5 mm2 pieces. Tissue samples were superficially disinfected by immersing in 70% alcohol for 10 sec, 2% sodium hypochlorite for 1 min, washed in sterile water, and placed on potato dextrose agar (PDA) at 28 ºC for 7 days in the dark. Twelve monosporic isolates derived from the PDA culture were morphologically characterized. Abundant white aerial mycelium and small pink to dark violet pigments in the center of the culture were observed. From 10-day-old cultures grown on carnation leaf agar medium microconidia and macroconidia were produced. Microconidia were hyaline, had zero to two septa, and were oval or ellipsoidal, 4.6 to 14 x 1.8 to 4.2 μm (n = 40). The macroconidia were hyaline, were slightly curved with three to five septa, and measured from 26 to 69 x 3 to 6.1 μm (n = 40). No chlamydospores were observed. According to the morphological characteristics, the isolates were identified as Fusarium verticillioides (Leslie and Summerell, 2006). DNA was extracted from one isolate and the Translation Elongation Factor 1-α (EF1) gene was amplified and sequenced (O'Donnell et al. 2010). The sequence obtained from isolate FV3CARCULSIN with 645 base pairs was submitted to NCBI GenBank with accession number OQ262963. The BLAST search revealed 100% similarity with F. verticillioides isolate 13 (KM598773) (Lizárraga et al. 2015). Identification in FUSARIUM ID resulted in a 99.85% similarity with isolate F. verticillioides CBS 131389 (MN534047) (Yilmaz et al. 2021). A phylogenetic tree, made with sequences of the EF1 gene, revealed that FV3CARCULSIN was most closely related to F. verticillioides (100% bootstrap). Pathogenicity tests were carried out on safflower plants (cv. Oleico) grown in sterile vermiculite. Plants were inoculated with a conidial suspension (1 × 105 conidia/ml) obtained from FV3CARCULSIN grown on PDA for 7 days. A total of 45 plants were inoculated by drenching the roots with 20 ml of inoculum when the plants were 20 days old. Fifteen plants served as negative controls without inoculation. Plants were kept for 60 days in greenhouse conditions; however, after 45 days the plants began to die. The assay was conducted twice. Rotting and necrosis was observed in the roots of the plants. The pathogen was reisolated from the tissue of all the plants with symptoms and identified as F. verticillioides using morphological characteristics and EF1 sequences, completing Koch's postulates. No symptoms were observed in control plants after 60 days. This is the first report of root rot in safflower caused by F. verticillioides in Mexico. The fungus has been reported in maize (Figueroa et al. 2010), but it is unknown if it could be the same pathogen of safflower. Identification of the pathogen is important for implementing management methods to reduce yield losses and for additional studies on the impact of the disease on oil quality extracted from safflower seeds.