Picarbutrazox Effectiveness Added to a Seed Treatment Mixture for Management of Oomycetes that Impact Soybean in Ohio.

None of the current oomycota fungicides are effective towards all species of Phytophthora, Phytopythium, Globisporangium, and Pythium that affect soybean seed and seedlings in Ohio. Picarbutrazox is a new oomyceticide with a novel mode of action towards oomycete pathogens. Our objectives were to evaluate picarbutrazox to determine (i) baseline sensitivity (EC50) to 189 isolates of 29 species, (ii) the efficacy with a base seed treatment with three cultivars with different levels of resistance in 14 field environments; and (iii) if the rhizosphere microbiome was affected by the addition of the seed treatment on a moderately susceptible cultivar. The mycelial growth of all isolates was inhibited beginning at 0.001 µg, and the EC50 ranged from 0.0013 to 0.0483 µg of active ingredient (a.i.)/ml. The effect of seed treatment was significantly different for plant population and yield in eight of 14 and six of 12 environments, respectively. The addition of picarbutrazox at 1 and 2.5 g of a.i./100 kg seed to the base seed treatment compared to the base alone was associated with higher plant populations and yield in three and one environments, respectively. There was limited impact of the seed treatment mefenoxam 7.5 g of a.i. plus picarbutrazox 1 g of a.i./100 kg seed on the oomycetes detected in the rhizosphere of soybean seedlings collected at the V1 growth stage. Picarbutrazox has efficacy towards a wider range of oomycetes that cause disease on soybean, and this will be another oomyceticide tool to combat early season damping-off in areas where environmental conditions highly favor disease development.

Host Range Characterization of Phytophthora sansomeana Across Corn, Soybean, Wheat, Winter Cereal Rye, Dry Bean, and Oats and an In Vitro Assessment of Seed Treatment Sensitivity.

Formally described in 2009, Phytophthora sansomeana is a pathogen of increasing interest in native, agricultural, and horticulturally important plant species. The objective of this study was to elucidate the symptomatic and asymptomatic host range of P. sansomeana on six agricultural crop species commonly used in field crop rotations in Michigan. In addition, sensitivity to oomicides commonly used in seed treatments, including oxathiapiprolin, mefenoxam, ethaboxam, and pyraclostrobin, was performed to aid in disease management recommendations. Plant biomass, quantity of P. sansomeana DNA in roots, and reisolations were used to assess pathogenicity and virulence of 18 isolates of P. sansomeana on each plant species using an inoculated seedling growth chamber assay. Isolates displayed varying levels of virulence to the hosts tested. Reisolations were completed for each plant species tested, and varying quantities of P. sansomeana DNA were found within all plant species root samples. Corn, wheat, soybean, dry bean, and winter cereal rye plants were symptomatic hosts with significant reduction observed in the total plant biomass. No significant reduction in total plant biomass was observed in oats, and oat roots harbored the least amount of P. sansomeana DNA. No P. sansomeana isolates were insensitive to the oomicide compounds tested with mean absolute inhibition (EC50) values of fungicide required for 50% growth inhibition values of 7.8 × 10-2 µg/ml for mefenoxam, 1.13 × 10-1 µg/ml for ethaboxam, 2.6 × 10-2 µg/ml for oxathiapiprolin, and 3.04 × 10-1 µg/ml for pyraclostrobin. These results suggest that common crop rotations in Michigan may not be a viable option to reduce soilborne inoculum accumulation and oomicide seed treatments could be considered for early-season management of P. sansomeana.

No Common Candidate Genes for Resistance to Fusarium graminearum, F. proliferatum, F. sporotrichioides, and F. subglutinans in Soybean Accessions from Maturity Groups 0 and I: Findings from Genome-wide Association Mapping.

Seedling diseases and root rot, caused by species of Fusarium, can limit soybean (Glycine max L.) production in the United States. Currently, there are few commercially available cultivars resistant to Fusarium. This study was conducted to assess the resistance of soybean maturity group (MG) accessions from 0 and I to Fusarium proliferatum, F. sporotrichioides, and F. subglutinans, as well as to identify common quantitative trait loci (QTLs) for resistance to these pathogens, in addition to F. graminearum, using a genome-wide association study (GWAS). A total of 155, 91, and 48 accessions from the United States Department of Agriculture (USDA) soybean germplasm collection from MG 0 and I were screened with a single isolate each of F. proliferatum, F. sporotrichioides, and F. subglutinans, respectively, using the inoculum layer inoculation method in the greenhouse. The disease severity was assessed 21 days postinoculation and analyzed using nonparametric statistics to determine the relative treatment effects (RTEs). Eleven and seven accessions showed significantly lower RTEs when inoculated with F. proliferatum and F. subglutinans, respectively, compared with the susceptible cultivar 'Williams 82'. One accession was significantly less susceptible to both F. proliferatum and F. subglutinans. The GWAS conducted with 41,985 single-nucleotide markers identified one QTL associated with resistance to both F. proliferatum and F. sporotrichioides, as well as another QTL for resistance to both F. subglutinans and F. graminearum. However, no common QTLs were identified for the four pathogens. The USDA accessions and QTLs identified in this study can be utilized to selectively breed resistance to multiple species of Fusarium.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

First report of Fusarium falciforme causing root and stem rot in cowpea (syn. black-eyed pea; Vigna unguiculata) in California.

In both April 2018 and September 2019, cowpeas / black-eyed peas (Vigna unguiculata) in one field in Tulare County, California were observed with tap root rot, both underground (foot) and aboveground stem rot, and in some cases canopy decline, compromising bean formation. In both fields, < 5% of plants appeared affected. Foot and stem segments (~1 cm) of 5-10 plants / field were disinfested sequentially with 0.1% Tween 20 (dip), 70% ethanol for 30 s, and 1% sodium hypochlorite for 2 min and placed on 1:10 potato dextrose agar with 0.03% tetracycline and Fusarium selective medium (Leslie and Summerell 2006). Fusarium-like isolates (dominant in isolation plates) were transferred to 0.6% KCl agar, where fusiform, curved macroconidia and varied microconidia in false heads on elongated monophialides were observed, characteristic of the Fusarium solani species complex (FSSC) (Leslie and Summerell 2006). Isolates CS221, CS222, and CS520 (representing different plants and locations) were saved as single hyphal tip cultures. An Illumina-derived genome sequence was assembled (Burkhardt et al. 2019) and partial tef1ɑ and rpb2 sequences (O'Donnell et al. 2022) were extracted from genome sequences in silico. Sequences were 99.9-100% identical to one another and to deposited F. falciforme isolates based on Fusarium ID and Fusarium MLST for tef1ɑ and rpb2, respectively (tef1a accessions: NRRL 28562 and NRRL 32331; rpb2 accession: NRRL 22857), and were deposited in GenBank (accessions in supplementary table). Pathogenicity was evaluated in three-week-old cowpea plants (cv. CB46rk2) in the greenhouse (13.5-33.6℃; 12:12 h L:D). The tap root / stem was wounded (1 mm wide, 2 mm deep) ~ 2 cm below the soil line and drenched with 50 ml of 106 spores / ml 0.1% water agar or with 0.1% water agar (negative control). The trial was arranged in a Randomized Complete Block Design with three blocks and 2-3 plants / isolate / block, and conducted twice. 52 d post-inoculation, below ground tap root / stem rot developed in 83% of F. falciforme-inoculated plants, with lesion lengths ranging from 25.2 ± 4.2 to 29.2 ± 8.0 mm (P = 0.893 for isolate, ANOVA). Canopy decline developed in 33-50% of plants across treatments in trial 1 (P = 0.859 for isolate) but not in trial 2, likely due to cooler conditions in trial 2 (January-March) vs. trial 1 (May-July), which were less stressful. F. falciforme isolates did not affect bean biomass (dry weight) vs. negative controls (12.5-14.8g / plant; P = 0.949 for pathogen treatment). FSSC isolates were recovered from 100% of symptomatic plants in the inoculated treatments but not in negative controls (both trials) and representative isolates from all treatments were confirmed as F. falciforme (tef1a analysis; trial 2 only). This study establishes F. falciforme as a root and stem rot pathogen of cowpea in California-a disease previously attributed to the morphologically and phylogenetically distinct F. phaseoli (syn. F. solani f. sp. phaseoli), but which lacked modern etiological studies (Frate et al. 2018; Geiser et al. 2021). This work is consistent with previous reports of F. falciforme as a root / stem rot pathogen in cowpea (Ajamu et al. 2023) and other beans (Sousa et al. 2017; Duarte et al. 2019). Clarification of disease etiology will improve accurate diagnosis and effective crop rotation-based management, since F. falciforme is also a pathogen of other California crops including melon, tomato and pistachio.

Characterization, Phylogenetic Analyses, and Pathogenicity of Eutiarosporella dactylidis on Sorghum in China.

Sorghum, the fifth-largest cereal crop globally and a C4 crop, mainly grows in arid and semi-arid areas. In 2021-2023, a new foliar disease of sorghum occurred in China. The diseased leaves showed water-soaked symptoms in the leaf tip and margins, resulting in half- and full-leaf desiccation and necrosis, thus affecting plant photosynthesis. A total of 24 Eutiarosporella strains were isolated from symptomatic leaves. Based on morphological characteristics and multi-locus phylogenetic analysis involving ITS, LSU, and EF1-α sequences, and the pathogenicity test, the pathogen of sorghum causing leaf blight in China was identified as Eutiarosporella dactylidis. The virulence of all E. dactylidis strains was evaluated using the spray-mycelium method. Different strains showed significantly different pathogenicities toward a susceptible cultivar, Longza 10, with disease indexes ranging from 23.76 to 60.37. This study first reported leaf blight of sorghum caused by E. dactylidis and named it "Eutiarosporella leaf blight", which provides a theoretical basis for farmers in disease management.

Chemical management strategies for halo blight of hop and in vitro sensitivity of Diaporthe humulicola populations to various fungicide classes.

Halo blight of hop, caused by Diaporthe humulicola, has increased in eastern North America since 2018. When left untreated, the disease can cause yield loss ranging from 17-56%. Currently, there are no fungicides registered for use on halo blight of hop. From 2020 to 2022 field trials were conducted using 10 fungicides registered for use on powdery and downy mildew of hop to determine their efficacy against halo blight. To validate field results, the EC50 value was determined for each active ingredient including flutriafol, tebuconazole + fluopyram, cyflufenamid, and trifloxystrobin + salicylhydroxamic acid (SHAM). Each fungicide tested had an EC50 value less than 50 ppm. A discriminatory dose was used to test the sensitivity of 206 D. humulicola isolates collected from the eastern U.S. and Canada in a poison agar assay. Results showed that tebuconazole + fluopyram decreased the incidence and severity of halo blight in the field. Also, this fungicide combination had EC50 values of 2.26 x 10-1 ppm and significantly reduced the growth of most of the isolates tested. Trifloxystrobin + SHAM decreased the presence of halo blight in the field tiral, but some isolates were less sensitive in discriminatory dose testing. Our results show that fungicides in FRAC groups 3, 7, and 11 were the most effective to control halo blight. Analyses of field trials showed a positive correlation between the severity of early season downy mildew infections and late season halo blight infections.

First Report of Alternaria alternata Causing Leaf Spot on Smooth Bromegrass (Bromus inermis Leyss.) in China.

Smooth bromegrass (Bromus inermis Leyss.) is an important forage crop in northern China. In July 2021, leaf spot symptoms were observed on smooth bromegrass in Ewenki Banner, Hulunbuir, Inner Mongolia. In an area of approximately 0.12 hectares, 95% disease incidence was observed. Ten diseased plants were collected for pathogen isolation. Leaf tissues near the lesions were cut into 5 × 5 mm pieces, surface-disinfested in 75% ethanol for 3 min, and rinsed with sterile distilled water. The pieces were placed on water agar in petri plates and incubated at 25℃ for three days. The resulting colonies were flushed with sterile water and a spore suspension was serially diluted and plated on potato dextrose agar (PDA). A single-spore colony was obtained. Ten isolates were obtained and designated HE1 to HE10. The colony morphology was identical for all isolates, grayish white in color on the upper surface and light black on the underside. The mycelia were light gray and velvety. Conidia were light brown to brown in color and oblate, oblong or oval. The conidial dimensions were typically between 15 to 43 µm by 8 to 9 µm in size. The conidia possessed one to six transverse septa, with slight to distinct constrictions at each division, and zero to two longitudinal septa. These morphological characteristics resembled Alternaria alternata (Fr.) Keissl.. DNA was extracted from three isolates, HE3, HE4 and HE5, using the CTAB method. Polymerase chain reaction (PCR) was performed on the extracted DNA with a set of primers ITS1/ITS4, H31a/H31b, gpd1/gpd2, TEF1-728F/TEF1-986R, and RPB2-5F2/fRPB2-7cR. The amplicon sequences from the three isolates were analyzed using the BLAST in GenBank (https://www.ncbi.nlm.nih.gov/). The results showed a high sequence identity, ranging from 99 to 100%, with the A. alternata strain YTMZ-20-2 across all the genetic markers tested. The strong match reinforced the identification of the strains as A. alternata. The sequences were deposited in GenBank (Table S1). The three fungal isolates were identified as A. alternata based on their morphological and genetic data. To conduct Koch's postulates, the representative isolate HE4 was used. Smooth bromegrass seed was soaked in water for four days and sown in potting soil contained in plastic pots (10 cm diameter × 15 cm height, five seeds/pot) in a greenhouse under a 16-h photoperiod at temperatures between 20 to 25°C and 60% relative humidity. When the plants reached a height of approximately 20 cm, the plants in three pots (replicates) were sprayed with a spore suspension (106 conidial/ml) at 10 ml/pot, and three pots were sprayed with sterile water for control. Five days after inoculation, the plants exhibited leaf spot symptoms similar to those previously described, while the control plants remained unaffected. The causative fungus was successfully re-isolated from the diseased plants and confirmed morphologically and molecularly on its identity as described above. This experiment was independently conducted three times. This is the first report of A. alternata causing leaf spot on smooth bromegrass in China. Since there is risk that the disease could seriously reduce the yield of the forage crop smooth bromegrass, further research is needed.

First Report of Wilt Caused by Fusarium nanum (FIESC 25) on Mungbean (Vigna radiata) in Pakistan.

Mungbean, Vigna radia (L.) R. Wilczek, is ranked 2nd next to chickpea (Cicer arietinum) in total cultivation and production in Pakistan. In August of 2022 and 2023, mungbean plants (cv. PRI Mung-2018) were found wilting in a field at the Ayub Agricultural Research Institute, Faisalabad, Pakistan. Wilted leaves turned yellow, died, but remained attached to the stem. Vascular tissue at the base of the stem showed light to dark brown discoloration. Roots were stunted with purplish brown to black discoloration. Symptomatic mungbean plants were collected from fields at five different locations (20 samples/location). Disease incidence was similar among the five fields, ranging from 5 to 10% at each location depending upon type of germplasm and date of sowing. For fungal isolation and morphological identification, symptomatic stem and root tissues were cut into ~5 mm2 pieces with a sterilized blade. Tissues were surface-sterilized for one min in a 0.5% sodium hypochlorite solution, rinsed twice in sterilized water, air dried on sterilized filter paper, and aseptically placed on potato dextrose agar (PDA) containing 0.5 g/L-1 streptomycin sulphate. Plates were incubated for 3-4 days at 25 ± 2°C with a 12-h photoperiod. Single-spore cultures were used for morphological and molecular analyses. Isolates on PDA grew rapidly and produced abundant white aerial mycelium that turned off-white to beige with age. Macroconidia were hyaline, falcate, typically 3-to-6 septate with a pointed apical cell and a foot-shaped basal cell, measuring 24.5-49.5 x 2.7-4.7 µm (n = 40). Globose to obovate chlamydospores measuring 5.8 ± 0.5 µm (n = 40) were produced singly or in chains and were intercalary or terminal and possessed roughened walls. The morphological data indicated the isolates were members of the genus Fusarium (Leslie and Summerell 2006). To obtain a species-level identification, a portion of translation elongation factor 1-α (TEF1), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2) region were PCR amplified and sequenced using EF1/EF2 (O'Donnell et al. 1998), Fa/G2R (Hofstetter et al. 2007), and 5f2/7cr (Liu et al. 1999) primers, respectively. DNA sequences of these genes were deposited in GenBank under accession numbers MW059021, MW059017 and MW059019, respectively. The partial TEF1, RPB1 and RPB2 sequences were queried against the Fusarium MLST database (https://fusarium.mycobank.org/page/Fusarium_identification), using the polyphasic identification tool. The BLASTn search revealed 99.9% identity of the isolate to F. nanum (Xia et al. 2019), formerly FIESC 25 of the F. incarnatum-equiseti species complex (MRC 2610, NRRL 54143; O'Donnell et al. 2018). To confirm pathogenicity, roots of 3-5 leaf stage mungbean seedlings were soaked in a 106 spores ml-1 conidial suspension of the fungus for 15 min and then planted in 10 cm pots containing sterilized soil. Mock-inoculated plants with sterile water served as a negative control. Twenty pots that were used for each inoculated and control treatment were maintained at 25 ± 2°C, 14:8 h photoperiod, and 80% relative humidity in a growth chamber. After 15 days, leaf yellowing, internal browning from the base of stems and root discoloration was observed in all the inoculated plants. The uninoculated negative control plants remained asymptomatic. Fusarium nanum was re-isolated from artificially inoculated plants and identified by colony growth, conidial characteristics on PDA and molecular analyses (TEF1). To our knowledge, this is the first report of wilt caused by F.nanum on mungbean in Pakistan. In Pakistan, mungbean cultivation in irrigated areas has increased in recent years. It has been introduced frequently in citrus orchards, crop rotation of maize and sesame, intercropping with sugarcane and as green manure. However, citrus, maize, sesame and sugarcane are also hosts of Fusarium spp. Therefore, this information warrants sustainable crop protection and may have an impact on further interaction of F. nanum with other wilt pathogens.

First Report of Clonostachys rosea Causing Bulb Rot on Fritillaria taipaiensis in China.

Taibai Beimu (Fritillaria taipaiensis) is a species of Fritillaria commonly used in traditional Chinese medicine for its antitussive, expectorant, and antihypertensive properties. In April of 2021 and 2022, an incidence 10-30% of yellowing or purpling, wilting, and dying symptoms was observed on Taibai Beimu in Wanyuan, Sichuan province. Infected roots and bulbs displayed spots ranging from brown to black, along with necrotic rot. In severe cases, the entire bulbs rotted. Fifteen symptomatic bulbs were cut into 0.5 × 0.5 cm pieces, surface sterilized in 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min under aseptic conditions, rinsed with sterile water 3 times, and air-dried. The segments were placed on potato dextrose agar (PDA) and incubated at 25℃ for 7 days in the dark. Six Clonostachys-like monospore isolates were obtained. Colonies on PDA reached 32 to 43 mm in diameter in 7 days at 25℃ in the dark, felty to tomentose to granulose aerial mycelia with a white or light yellow appearance, and reverse colors matching. On cornmeal-dextrose agar, primary conidiophores had a Verticillium-like structure with 1 to 3 levels. Stipes were 36.1 to 236.3µm long. Phialides formed in whorls of 2 to 5, 15.3 to 45.7µm long, 1.1 to 3.4µm wide at the base, and 1.03 to 2.41µm wide near opening (n=95). Each producing a small hyaline drop of conidia. Conidia were 3.7 to 11.3µm × 2.1 to 4.1µm (n=110). Secondary conidiophores displayed Penicillium-like structures, and stipes were 23.1 to 142.3µm long. Phialides formed in compressed whorls of 4 to 8 per metula, 7.0 to 16.0µm in length, 1.3 to 3.1µm in width at the base, 1.8 to 3.6µm at the widest point, and 0.8 to 1.8µm near opening (n=50). Conidia were 3.0 to 6.4µm ×1.6 to 3.4µm (n=65). The morphology was consistent with the previous description of Clonostachys rosea (Hans-Josef et al. 1999). The ATP citrate lyase (ACL1), ß-tubulin (TUB2), translation elongation factor 1-α (tef1α), and the nuclear ribosomal internal transcribed spacer (ITS) of three strains were amplified and sequenced using primers acl1-230up/acl1-1220low (Gräfenhan et al. 2011), T1/CYLTUB1R (Crous et al. 2004; O'Donnell and Cigelnik 1997), EF1-728F/EF2 (Carbone and Kohn 1999; O'Donnell et al. 1998), and ITS1/ITS4 (White et al. 1990), respectively. Blastn homology search showed a > 97% similarity to the ex-type strains of C. rosea (CBS710.86). All sequences have been deposited in GenBank (PP394342 to PP394350, and PP396901 to PP396903). A phylogenetic tree was constructed using Bayesian analysis based on the alignment of the combined ACL1, TUB2, tef1α, and ITS sequences through IQ-TREE. The tree displayed clustering with known strains of C. rosea. Pathogenicity was confirmed by inoculating five healthy five-year-old Taibai beimu plants with a spore suspension (1.0 × 106 spores mL-1) of the strain WYEB1101, while sterilized water was used as a control. The inoculation process involved pouring the spore suspension over the wounded bulbs and covering with them sterile soil. Subsequently, all plants were cultivated in sterile soil indoors under natural conditions suitable for Taibai beimu. The pathogenicity assays were repeated twice. After 20 days of cultivation, the infected plants displayed symptoms similar to those observed in the field, while all control plants remained asymptomatic. Sequencing confirmed the re-isolation of C. rosea from the inoculated plants, satisfying Koch's hypothesis. Clonostachys rosea has been previously reported to cause root rot of Chinese medicine herb, such as Astragalus membranaceus and Gastrodia elata (Lee et al. 2020; Qi et al. 2022). To our knowledge, this is the first report of C. rosea infecting Taibai Beimu in China, highlighting a potential risk to this crop.

Discovery of Fusarium pseudograminearum Causing Crown Rot of Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Fusarium pseudograminearum is an important plant pathogen that invades many crops (Zhang et al. 2018). Since it was first discovered in Australia in 1951, F. pseudograminearum has been reported in many countries and regions and caused huge economic losses (Burgess et al. 2001). In 2012, crown rot of wheat caused by F. pseudograminearum was discovered for the first time in Henan Province, China (Li et al. 2012). Wheat (Triticum aestivum L.) is one of the most important food crops in Xinjiang Uygur Autonomous Region (XUAR), with 1.07 million hectares cultivated in 2020. In June 2023, a survey of crown rot disease was carried out in winter wheat cv. Xindong 20 in Hotan area, XUAR, China (80.148907°E, 37.051474°N). About 5% of wheat plants showed symptoms of crown rot such as browning of the stem base and white head. The disease was observed in 85% of wheat fields. In order to identify the pathogens, 36 pieces of diseased stem basal tissue, 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, then rinsed three times with sterile water and placed on potato dextrose agar (PDA) medium at 25°C. A total of 27 isolates with consistent morphological characteristics were obtained using single-spore technique (Leslie and Summerell. 2006), and the isolation rate was 75%. The isolates grew rapidly on PDA, produced large numbers of fluffy white hyphae, and pink pigment accumulated in the medium. The isolates were grown on 2% mung bean flour medium and identified by morphological and molecular methods. Macroconidia were abundant, relatively slender, curved to almost straight, commonly two to seven septate, and averaged 22 to 72 × 1.8 to 4.9 µm. Microconidia were not observed. The morphological characters are consistent with Fusarium (Aoki and O'Donnell. 1999). Two isolates (LP-1 and LP-3) were selected for molecular identification. Primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') were used to amplify a portion of the EF-1α gene (O'Donnell et al. 1998). The two 696 bp PCR products were sequenced and submitted to GenBank. The EF-1α gene sequences (GenBank Accession No: PP062794 and PP062795) shared 99.9% identity (695/696) with published F.pseudograminearum sequences (e.g., OP105187, OP105184, OP105179, OP105173). The identification was further confirmed by F. pseudograminearum species-specific PCR primers Fp1-1/Fp1-2 (Aoki and O'Donnell. 1999). The expected PCR products of 518 bp were produced only in F. pseudograminearum. Pathogenicity tests of LP-1 and LP-3 isolates were performed on 7-day-old seedlings of winter wheat cv. Xindong 20 using the drip inoculation method with a 10-µl of a 106 macroconidia ml-1 suspension near the stem base (Xu et al. 2017). The experiment was repeated five times in a 20 to 25°C greenhouse. Control seedlings were treated with sterile water. After 4 weeks, wheat seedling death and crown browning occurred in the inoculated plants with over 90% incidence. No symptoms were observed in the control plants. The pathogen was reisolated from the inoculated plants by the method described above and identified by morphological and PCR amplification using F. pseudograminearum species-specific primers Fp1-1/Fp1-2. No F. pseudograminearum was isolated from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of winter wheat in XUAR of China. Since F. pseudograminearum can cause great damage to wheat, one of the most important food crops in China, necessary measures should be taken to prevent the spread of F. pseudograminearum to other regions.

Gray mold disease on bottle gourd caused by Cladosporium tenuissimum in China.

Bottle gourd [Lagenaria siceraria (Mol.) Stand] is a widely cultivated succulent crop species. In December 2022, a serious bottle gourd disease occurred in the protected vegetable planting base of Xingguo County, Ganzhou City, Jiangxi Province, China, with 85% of the 2,100 plants having gray mold disease-like symptoms, including gray spots on the infected fruit. They quickly expanded at suitable temperature and humidity, forming a gray mold layer with inward depressions, which spread to the fruit stem causing watery rot, and the flesh turned black and started to rot. To isolate the pathogen, fruits of the diseased plants were surface-disinfected with 75% ethanol for 30 s, immersed in 0.1% HgCl2 for 1 min, rinsed thrice with sterile water, and cultured on a potato-dextrose agar (PDA) medium at 28°C. Mycelia from the diseased tissue were subcultured on fresh PDA medium to obtain pure cultures. After incubation at 25°C for 7 days, olive-green colonies (~2.5 mm·d-1) developed. Cultures developed numerous elliptical and limoniform conidia measuring 2.69~9.79 µm to 2.10~5.92 µm (average 5.62×3.12 µm) (n=20). The morphological characteristics of the pathogen resembled those of Cladosporium spp. Fungal genomic DNA was extracted, and the internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (TEF-1α), and actin (ACT) regions were amplified with primers ITS1/4, TEF-728F/986R, and ACT-512F/783R, respectively, and sequenced (Bensch et al. 2012; Jo et al. 2018). Basic Local Alignment Search Tool analysis (BLAST) revealed that the ITS (accession no. OQ186729), ACT (OQ240962), and TEF-1α (OQ240963) sequences of isolate hjt4 shared the highest similarity (99-100%) with those of Cladosporium tenuissimum (accessions no. OM232068, OM256530, OM256526) (Duccio et al. 2015). A phylogenetic tree of the isolate hjt4 and its close relatives within Cladosporium was constructed using the MEGA X neighbor-joining method. The pathogen was identified as C. tenuissimum based on morphological and molecular characteristics. A specimen (JXAU-H2022982) was deposited at the Herbarium of the College of Agronomy, Jiangxi Agricultural University. To confirm its pathogenicity, seven-day-old healthy bottle gourd fruits were disinfected with 75% ethanol, 1 mm-deep wounds were made with sterilized scalpels, and the plants were inoculated with PDA plugs (0.8 cm in diameter) containing actively growing mycelia of isolate hjt4. Plants inoculated with sterile PDA plugs served as controls. Each group contained three fruits, and the experiment was performed in triplicate. All fruits were incubated in a biochemical incubator at 28°C. After 3 days, the fruit surface shrank, and the flesh turned to a black colour and rotten, which rapidly spread to the branches. Control fruits did not develop any symptoms. Reisolated colonies showed the same morphological traits as those of the inoculation isolates, whereas no target colonies were isolated from the control fruits. The pathogen was previously reported to cause leaf blight disease in Coriandrum sativum (Zhou et al. 2022) and sooty spots on Cape gooseberry (Miyake et al. 2022), among others. To our knowledge, this is the first report of gray mold disease caused by C. tenuissimum on bottle gourd in China. The findings provide an important foundation for monitoring and controlling the spread of this disease.

First report of Cassava Bacterial Blight caused by Xanthomonas phaseoli pv. manihotis in the Amazonian forest of Ecuador.

Xanthomonas phaseoli pv. manihotis (Xpm) is a plant pathogenic bacterium known as the causal agent of cassava bacterial blight (CBB). CBB is the most limiting bacterial disease affecting cassava (Manihot esculenta Crantz), characterized by diverse symptoms including angular water-soaked leaf lesions, blight, wilting, stem exudates, stem cankers and dieback. CBB has been reported in most cassava-growing regions around the world, and, under conducive conditions, crop yield losses can reach up to 100% (Zárate-Chaves et al. 2021). While Xpm genetic diversity is remarkably high in South America (Bart et al. 2012) and cassava originates and was domesticated in the Amazon basin (Allem 2002), reports of CBB in the Amazonian region are missing. To fill this gap, in October 2018 we surveyed for CBB symptoms in cassava fields of the Orellana Province, located in the Amazon forest of the Republic of Ecuador. Adult cassava plants exhibiting typical angular, water-soaked leaf lesions were found in polyculture plots, i.e. intercrops of cassava with other species such as plantains and fruit trees (a.k.a. chakras). After surface disinfection with 5% sodium hypochlorite followed by 70% ethanol, white Xpm-like colonies were isolated from diseased leaf tissues of four plants on YPGA medium (yeast extract, 5 g/l; peptone, 5 g/l; glucose, 5 g/l; agar-agar, 15 g/l) supplemented with cephalexin (40 mg/l) and cycloheximide (50 mg/l). Pathogenicity tests were performed on peat-potted, 2-month-old cassava plants of the cultivar 60444. Bacterial suspensions were adjusted to an OD600 of 0.2 (2 × 108 CFU/ml) in sterile 10-mM MgCl2 and syringe infiltrated in fully-expanded leaves. In parallel, 20 µl of each bacterial suspension adjusted to an OD600 of 0.02 (2 × 107 CFU/ml) were inoculated on stems inside a hole previously punched with a sterile needle in the junction of the third-top petiole. Sterile 10-mM MgCl2 was used for mock inoculations in both leaves and stems, and experiments were replicated in three plants. Plants were incubated in a greenhouse at 28 ± 1°C with a 12-h photoperiod. Infiltrated leaves developed watersoaking 3 days post inoculation, while wilted leaves, stem exudates, and dieback were observed 21 days after stem inoculation. Control plants remained symptomless. White Xpm-like colonies were re-isolated from symptomatic leaves (Fig S1). One colony of each of the four Xpm isolates (before and after re-isolation) was assessed using diagnostic PCRs (Bernal-Galeano et al. 2018; Flores et al. 2019), using strain Xam668 as positive control. All four candidates were positive for both diagnostic tools. The sequences of the housekeeping genes atpD, dnaK, efp, glnA, gyrB and rpoD of our isolates were extracted from full genome sequences obtained through Oxford Nanopore Technologies (ONT) (GenBank OR288194 to OR288217) and compared to their homologs in four close Xanthomonas species and a reference Xpm strain (Table S1). The sequences of the tested strains aligned with that of Xpm CIO151 (GCA_004025275.1) (Arrieta-Ortiz et al. 2013) with nucleotide identity above 99.92% (Fig S2). The four strains were named CIX4169, CIX4170, CIX4171 and CIX4172, stored in the IRD Collection of Xanthomonas, where they are available upon request. To our knowledge, this is the first report of CBB in the Amazonian region and in Ecuador, where cassava is a central element for local culture and economy. Further surveys will be necessary to evaluate the distribution and prevalence of CBB in other ecozones of Ecuador where cassava is cultivated.

First report of Stagonosporopsis cucurbitacearum Causing Gummy Stem Blight on Trichosanthes kirilowii Maxim. in China.

Trichosanthes kirilowii Maxim. (Cucurbitaceae), one of the Chinese herbal medicines, is an economically important crop in Anhui Province, China. In recent years, gummy stem blight disease, a major disease of cucurbits, was widespread in many T. kirilowii plantations. The initial symptoms on the naturally infected stems appeared as dark brown water-soaked lesions, and as the disease progressed, vines of T. kirilowii gradually withered. On leaves, brown water-soaked lesions were visible initially, and then lesions enlarged and coalesced, resulting in extensive necrosis of leaves. On fruit, lesions covered with the white mycelium were nearly circular and tan to brown initially. Subsequently, the diseased fruit turned black and rotten commonly known as fruit rot or black rot. A Stagonosporopsis-like organism was consistently isolated from symptomatic stems, leaves and fruits. Fungal isolates were initially white and later turned dark grey or black with woolly to floccose aerial mycelium on PDA medium. Twenty-four isolates from different plantations were selected for further morphological studies. Pycnidia and conidia were formed after inoculating on cucumber fruit for 3 days. Pycnidia were globose to sub-globose, brown, ostiolate and 106.7 to 213.6 µm (average 160.1 µm, n = 50) in diameter. Conidia were hyaline, ellipsoidal, aseptate or one-septate, slightly constricted at the septa, 6.1 to 13.6 × 3.5 to 4.8 µm (average 9.9 × 4.1 µm, n = 50), and contained two or more oil drops. Three different loci of the genomic DNA, including the nuclear ribosome DNA internal transcribed spacer (ITS), RNA polymerase II second-largest subunit (RPB2), and ß-tubulin (TUB2) genes., were amplified using primers ITS1/ITS4 (White et al. 1990), RBP2DF/RBP2DR (Lawrence et al. 2013), and T1/ß-Sandy-R (O' Donnell and Cigelnik 1997; Stukenbrock et al. 2012), respectively and sequenced. A phylogenetic tree was built based on analysis of ITS, RPB2, and TUB2 sequences that deposited in GenBank (MW485497-MW485502 for ITS, MW531661-MW531666 for RPB2, and MW531667-MW531672 for TUB2), using the maximum likelihood method. The phylogenetic tree showed that the isolates fell into a single clade with S. cucurbitacearum. On the basis of morphological and molecular characteristics, the isolates obtained from T. kirilowii were identified as Stagonosporopsis cucurbitacearum. Pathogenicity tests were carried out on stems and leaves of 4-week-old T. kirilowii seedlings and on immature fruit collected from adult T. kirilowii plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were similarly inoculated with agar plugs. The diameters of lesions were measured in two perpendicular directions. Re-isolations from the stem and leaf lesions were performed on the PDA medium. Stagonosporopsis cucurbitacearum, was re-identified based on its colony and conidial characteristics and, therefore, completed Koch's postulates. Gummy stem blight caused by S. cucurbitacearum has been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on T. kirilowii caused by S. cucurbitacearum in China. The research provides a basis for the development and implementation of effective management strategies. Pathogenicity tests were carried out on stems and leaves of 4-week-old T. kirilowii seedlings and on immature fruits collected from adult T. kirilowii plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were treated similarly but inoculated with agar plugs. Diameters of lesions were measured in two mutually perpendicular directions. Reisolations from the lesions were performed on PDA medium, and was re-identified based on its colony and conidial characteristics to complete Koch's postulates. Gummy stem blight caused by S. cucurbitacearum have been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on T. kirilowii caused by S. cucurbitacearum in China. The research provides a basis for the development and implementation of effective management strategies.

Occurrence of Fusarium Crown Rot Caused by Fusarium culmorum on Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Wheat (Triticum aestivum L.) is the predominant grain crop and plays a pivotal role in grain production in Xinjiang Uygur Autonomous Region (XUAR), China. Its cultivated area constitutes approximately half of the total sown area of grain crops in XUAR, with 1.14 million hectares in 2021. Fusarium crown rot (FCR) of wheat, caused by Fusarium culmorum (W.G. Smith) Sacc., is one of the most devastating soil-borne diseases known to seriously reduce grain yield (Ma et al. 2024; Saad et al. 2023). In 2016, FCR of wheat, caused by F. culmorum, was firstly identified in Henan Province, China (Li et al. 2016). In June 2023, during the investigation of FCR of wheat in Aksu Prefecture, XUAR, FCR on winter wheat (cv. Xindong 20) was found (82.761349°E, 41.612202°N). The grain-filling period for winter wheat in early June coincided with a period of high temperatures and water demand in Aksu Prefecture. Approximately 8% of the Xindong 20 wheat plants exhibited symptoms of white heads and browning at the stem base, with the disease present in 82% of the wheat fields surveyed. To identify the pathogens, 20 samples of diseased stem basal tissue, each 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, followed by three rinses with sterile water. These samples were then plated onto potato dextrose agar (PDA) medium at 25°C for 5 days. A total of 17 isolates with consistent morphological characteristics were obtained using single-spore technique, with an isolation rate of 85%. The isolated strains exhibited rapid growth on PDA, producing fluffy, pale-yellow hyphae, and accumulating a pale-yellow to dark red pigment on the bottom of the medium. On carnation leaf agar (CLA), these strains formed orange colonies due to the aggregation of a large number of macroconidia. The macroconidia were short and thick, with three to four septa and rounded apical cell, averaging 31.94 to 40.96 × 5.62 to 6.71 µm (Magnification of ×400). Microconidia were not observed. These morphological characters were consistent with those of F. culmorum (Leslie and Summerell. 2006). Two isolates (D-9 and D-11) were selected for molecular identification. The EF-1α gene fragment was amplified using primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') as previously described by O'Donnell et al. (1998). The two 665 bp PCR products were sequenced and submitted to GenBank (GenBank Accession No: PP763247 and PP763248) with 99. 7% identity to the published F. culmorum sequences (e.g., OP985478, OP985477, MG195126, KX702638). The molecular identification was further confirmed by F. culmorum species-specific PCR primers FcOIF/FcOIR (Nicholson et al. 1998). The expected PCR products of 553 bp were produced only in F. culmorum. Strains D-9 and D-11 were used to conduct the pathogenicity experiment on 7-day-old winter wheat (cv. Xindong 20) using drip in the lower stem inoculation method with a 10-µl of 106 macroconidia ml-1 suspension, and the control 7-day-old winter wheat were treated with sterile water (Xu et al. 2017). The experiments were replicated five times in a greenhouse at temperatures ranging from 20℃ to 25℃. After 4 weeks, all inoculated wheat seedlings showed stem base browning or even death. No symptoms were observed on the control plants. The fungus was reisolated from all inoculated wheat plants by the method described above and identified by morphological and PCR amplification using F. culmorum species-specific primers FcOIF/FcOIR. No F. culmorum was isolated from the control wheat plants, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F.culmorum causing FCR on winter wheat in XUAR, China. Considering wheat is the predominant grain crop and plays a pivotal role in grain production in China, necessary measures should be taken to prevent the spread of F. culmorum to other regions.

First report of Fusarium verticillioides causing leaf rot on Artemisia argyi in China.

Artemisia argyi is a perennial herb native to East Asia. It is an important traditional Chinese medicinal plant known for its strong flavor and medicinal effects. It is rich in active ingredients and has a wide range of biological activities, including anti-inflammatory, antioxidant, and immune regulation properties. From May to July in 2023, a serious leaf rot outbreak occurred on A. argyi in several farms (approximately 200 acres) in Tanghe county (32°46'44" N, 112°43'13" E), Henan Province, China. The incidence rate reached 65% (n=200). Pale yellow spots (1-2 cm in diameter) first appeared on the leaves, then expanded to form irregular yellowish-brown lesions, eventually causing the entire leaves to wither. Diseased leaves (30) were collected and cut into 5 x 5 mm2 pieces in the areas between infected and healthy tissues. The excised plant tissues were sterilized in 75% ethanol and 1% sodium hypochlorite solution for 30 seconds and one minute, respectively. The tissues were then rinsed with sterile water and placed on potato dextrose agar (PDA) followed by incubating at 25 °C for 3 days. The isolated strains belonged to the genera Fusarium and Alternaria. After pathogenicity verification, 25 purified Fusarium strains were obtained. Three representative strains (AC-Q, AC-X, AC-Y) from different regions were used for further studies. Each strain formed abundant aerial mycelium that was initially white and later developed into purple pigments. Aerial conidiophores were sparsely branched, terminating with verticillate phialides. Macroconidia were slender, straight, and measured 21.8 to 47.5 × 3.1 to 4.4 µm, with two to four septa. Microconidia were clavate and measured 8.31 to 11.6 × 2.1 to 3.5 µm. Morphological characteristics were consistent with the species description of Fusarium verticillioides (Sacc.) Nirenberg 1976 (Leslie and Summerell, 2006). The rDNA internal transcribed spacer (ITS), ß-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase II largest subunit (rpb1) and RNA polymerase II second largest subunit (rpb2) were amplified for molecular identification (O'Donnell et al., 2022). The sequences were deposited in GenBank with accession Nos. OR960548, OR960552, OR960555 (ITS), OR972413, OR972414, OR972415 (tub2), OR797685, OR797686, OR797687 (tef1), OR972410, OR972411, OR972412 (cmdA), PP035106, PP035107, PP035108 (rpb1), and PP035109, PP035110, PP035111 (rpb2). BLASTn analysis of AC-Q sequences exhibited 99 to 100% similarity with F. verticillioides sequences (strains CBS 576.78) MT010888 of cmdA, MT0109566 of rpb1, and MT010972 of rpb2. A phylogenetic tree was constructed with concatenated sequences (tub2, tef1, cmdA, rpb1, rpb2), alongside the sequences of the type strains using the neighbor-joining method. The three strains formed a clade with the type strain CBS 576.78 of F. verticillioides, and were separated from other Fusarium spp. These morphological and molecular identifications indicated that the pathogen was F. verticillioides. Pathogenicity was tested on 10 healthy 2-month-old potted seedlings by spraying them with a conidial suspension (106 conidia ml-1), and 5 seedlings were sprayed with sterilized water as a control. The plants were placed in a climate incubator at 28°C and a relative humidity of approximately 90%. Ten days after seedling inoculation, typical lesions were observed on the treated plants, except in the control group. The reisolated strains were identified as F. verticillioides by morphological and molecular characterization, fulfilling Koch's postulates. F. verticillioides is known to cause Fusarium ear rot on maize, as well as diseases on other plants in China such as Brassica rapa (Akram et al., 2020) and Schizonepeta tenuifolia (Li et al., 2024). This is the first report of F. verticillioides causing leaf rot on A. argyi worldwide. Identification of the pathogen is crucial for implementing management approaches to reduce yield losses.

First Report of Xanthomonas euvesicatoria pv. sesami Causing Leaf Spot on Sesame (Sesamum indicum L.) in South Carolina, USA.

Sesame (Sesamum indicum L.) is an annual plant known as one of the first domesticated oilseed crops. It is cultivated worldwide, mostly in Asia, Africa, and the Americas (Singh, 2006). In August 2022 and September 2023, dark angular necrotic spots on leaves and stems (100% incidence), blights, and severe defoliation were observed in a 4-acre rainfed sesame field located in the Colleton County of South Carolina, USA (Fig. S1). Bacterial streaming from cut leaf lesions was observed from diseased plants in both years. Two plants were collected for pathogen isolation in 2023. Symptomatic leaves were surface sterilized with 70% ethanol for 1 min and dried in a laminar flow hood. For each isolate, four sterile toothpicks were used to poke lesion margins and stirred in 300 µl of sterile distilled water in a 2-ml sterile microcentrifuge tube and soaked at room temperature (c. 21 °C) for 10 min. Each bacterial suspension (10 µl) was streaked on nutrient agar (NA) in a Petri dish. Convex and mucoid yellow colonies formed after a 48-h incubation at 28°C in the dark. Two isolates (S813 and S814), one from each plant, were obtained by transferring single colonies to new NA plates. Both isolates were preliminarily identified as Xanthomonas [S813: X. campestris (P = 0.53); S814: X. campestris (P = 0.77)] using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of the atpD and dnaK genes was performed for both isolates using the conditions described in Félix-Gastélum et al. (2019). The sequences of both amplicons are 100% identical for each gene between the two isolates. PCR and sequencing of the gyrB gene was also done for S813 with the primers from Young et al. (2008). The atpD (S813/S814), dnaK (S813/S814), and gyrB (S813) sequences (GenBank accessions: PP507118 to PP507120) showed the best match with 100% identity to the corresponding gene sequences [GenBank accessions: KJ491167 (100% coverage), KJ491257 (99% coverage), EU285201 (100% coverage)] of the X. euvesicatoria pv. sesami (=X. campestris pv. sesami) type strain LMG865 (Constantin et al. 2015, Parkinson et al. 2009). A neighbor joining tree with the concatenated sequences of these three genes (2,210 nt) showed that S813 and LMG 865 had the closet relationship with X. euvesicatoria pv. alfalfae (CFBP3836, Fig. S2). To fulfill Koch's postulates, three healthy sesame plants (cultivar Shirogoma) were spray inoculated separately with each suspension of S813 and S814 in sterile tap water until runoff (approx. 5×108 CFU/ml). Two sesame plants were sprayed with sterile tap water and served as negative control. All plants were maintained in a greenhouse at approximately 28/20°C (day/night) with natural photoperiod. Dark leaf spots and leaf yellowing were observed on inoculated plants 7 to 14 days after inoculation. No disease symptom was observed on the control plants. Bacteria were reisolated from leaf spots of the inoculated plants and confirmed to be X. euvesicatoria pv. sesami based on atpD and dnaK sequences. The disease was first reported in Sudan (Sabet and Dowson, 1960), after which it was reported in USA (Isakeit et al., 2012) and Mexico (Félix-Gastélum et al. 2019). To the best of our knowledge, this is the first report of this disease in South Carolina, USA. Since the interest of sesame to the farmers is increasing in the southeastern USA, it is necessary to perform further research to examine the disease distribution and its economic impact.

Biological Control of Rapeseed Clubroot (Plasmodiophora brassicae) Using the Endophytic Fungus Didymella macrostoma P2.

Didymella macrostoma P2 was isolated from rapeseed (Brassica napus), and it is an endophyte of rapeseed and an antagonist of three rapeseed pathogens, Botrytis cinerea, Leptosphaeria biglobosa, and Sclerotinia sclerotiorum. However, whether P2 has a suppressive effect on infection of rapeseed by the clubroot pathogen Plasmodiophora brassicae remains unknown. This study was conducted to detect production of antimicrobials by P2 and to determine the efficacy of the antimicrobials and P2 pycnidiospores in suppression of rapeseed clubroot. The results showed that cultural filtrates (CFs) of P2 in potato dextrose broth and the substances in pycnidiospore mucilages exuded from P2 pycnidia were inhibitory to P. brassicae. In the indoor experiment, seeds of the susceptible rapeseed cultivar Zhongshuang No. 9 treated with P2 CF and the P2 pycnidiospore suspension (P2 SS, 1 × 107 spores/ml) reduced clubroot severity by 31 to 70% on the 30-day-old seedlings compared with the control (seeds treated with water). P2 was reisolated from the roots of the seedlings in the treatment of P2 SS; the average isolation frequency in the healthy roots (26%) was much higher than that (5%) in the diseased roots. In the field experiment, seeds of another susceptible rapeseed cultivar, Huayouza 50 (HYZ50), treated with P2 CF, P2 CE (chloroform extract of P2 CF, 30 µg/ml), and P2 SS reduced clubroot severity by 29 to 48% on 60-day-old seedlings and by 28 to 59% on adult plants (220 days old) compared with the control treatment. The three P2 treatments on HYZ50 produced significantly (P < 0.05) higher seed yield than the control treatment on this rapeseed cultivar, and they even generated seed yield similar to that produced by the resistant rapeseed cultivar Shengguang 165R in one of the two seasons. These results suggest that D. macrostoma P2 is an effective biocontrol agent against rapeseed clubroot.

First report of Curvularia spicifera (≡ Bipolaris spicifera) causing spathe blight and leaf spot disease of pearl millet (Cenchrus americanus) in India.

Cenchrus americanus (L.) Morrone (Poaceae), is an important millet crop cultivated mainly in arid and semiarid regions and is a staple food grain for millions of people. During 2021 July surveys in the pearl millet fields in Mysore (12°30'55" N; 76°56'54" E), Karnataka, India, plants showed spathe blight and leaf spot disease with an overall incidence ranging from 5 - 8% in the 15 hectares surveyed. Infected leaves appeared brown, and lesions extended to the sheath. Some spathes were also found infected with similar symptoms. Diseased leaves and spathes were collected (n = 5 each) for pathogen identification. Samples were cut into small pieces (0.5 cm2), sterilized with sodium hypochlorite (2%, v/v), and blotted dried. The associated fungal pathogen was isolated on potato dextrose agar (PDA) medium amended with Streptomycin (40 mg/L) and incubated at 28 ºC for 1 week. Colonies were grey, fluffy, cottony with an irregular margin, undulate and dark brown in the back of the plate. Conidiophores were pale brown, erect, slightly curved, septate, unbranched, verruculose and measured 27.1 - 94 µm in length × 2.3 - 4.5 µm in width (n = 20). Conidiogenous cells were brown, subcylindrical, irregularly shaped, and conidia were straight, mainly elliptical, dark brown smooth, with two to three septa, with measurements of 11.1 - 26.4 µm by 5.7 - 14.3 µm (n = 50). Based on morphological characters, the pathogen was identified as Curvularia sp. Two representative isolates (UOMPM1 & UOMPM2) were molecularly identified. The total genomic DNA was extracted with a CTAB method, and ITS, GAPDH and tef-1α loci were amplified using primers ITS1/ITS4 (White et al., 1990), GPD1/GPD2 (Berbee et al., 1999) and EF1983F/EF-2218R (Schoch et al., 2009) respectively. ITS sequence had 100% similarity (706/706bp) with reference sequence C. spicifera (MH863648; HF934915 & HF934916); tef-1α sequence had 100% (933/933bp) identity with C. spicifera (KM062878, KJ939505), and the GAPDH sequence was 99.8% identical to that of Curvularia sp. (MG979055), and C. spicifera (MH809681). Combined dataset of concatenated sequence (ITS-GAPDH-tef-1α) was used in a phylogenetic analysis and revealed that the isolates were in a common clade with the isolate of Curvularia spicifera (CBS 274.52) thus, confirming the identity of the isolated pathogen as C. spicifera. The sequences obtained in the present study were deposited in the GenBank (ITS: OQ253406, OQ253407; LSU: OQ253429, OQ253430; GAPDH: OQ263372, OQ263373 & TEF: OQ263374, OQ263375). Pathogenicity test was carried out by inoculating (foliar /whole plant spray) 60 healthy pearl millet plants (45-days old), grown in field plot with spore suspension (105 conidia/ml). Control plants (n=20) were treated with sterile water. The experiments were conducted in triplicates and repeated twice. Development of disease symptoms was recorded on 41 plants, and all control plants remained healthy. The identity was confirmed after re-isolation as C. spicifera based on cultural and molecular sequence analysis. To our knowledge, this is the first report of C. spicifera causing a leaf spot and spathe blight disease of pearl millet in India. This disease seriously affects grain production, and effective disease management strategies need to be investigated.

First Report of Basal Stem Rot of Achnatherum inebrians Caused by Fusarium pseudograminearum in China.

Drunken horse grass (Achnatherum inebrians) is a perennial bunchgrass that is widely distributed in arid and semi-arid grasslands in northwest China (Zhang et al., 2021). In July 2023, Basal stem rot was found in artificially grown drunken horse grass plots in Yuzhong County (35.85° N, 104.12° E), Gansu Province, China, with an average incidence of 5.2%. Diseased plants showed crown and basal stem rot with chocolate brown discoloration at the base of the stem and slight constriction of some basal stems. Five field's foci were surveyed and at least 6 basal stems per focus were collected. Infected basal stems were surface-sterilized (75% ethanol for 30 s and 1% NaClO for 90 s), rinsed three times with sterilized water, placed on potato dextrose agar (PDA), and incubated at 22°C in the dark for 3 days. Isolates were purified by single spore cultures (Leslie and Summerell, 2006). The average mycelial growth rate was 4.8 to 7.5 mm/day at 25°C on PDA, and the colonies produced aerial mycelium varying from rose to yellow white, and rose to burgundy pigment diffused into the agar. Macroconidia of the isolates were produced on carnation leaf agar (CLA) incubated under black light and observed to be abundant, but no microconidia were found. Macroconidia were relatively slender, curved to almost straight, commonly 3-6 septate, averaging 30.1 × 3.8 µm (n=50). The morphological characteristics of this fungus fully fit the description of F. pseudograminearum (Aoki and O'Donnell, 1999). To obtain the phylogenetic support, DNA of three representative isolates YZ-Y-1, YZ-Y-2 and YZ-Y-3 was extracted by using an HP Fungal DNA Kit (D3195), and a portion of the RNA polymerase II second largest subunit (RPB2) gene and elongation factor 1 alpha (EF1-α) gene were amplified using primers RPB2-5f2 and RPB2-7cr (O'Donnell et al. 2010) and EF1 and EF2 (O'Donnell et al. 1998), respectively. Results of sequences were deposited in GenBank (accession nos. PP179044 to PP179049). A nucleotide BLAST search revealed RPB2 and EF1-α sequences to be 99.8 and 100% similar to the corresponding sequences of the ex-type strain NRRL 28062 F. pseudograminearum accessions numbers MW233433 and MW233090, respectively. For pathogenicity tests, 15 µl of conidia suspension (1×106 conidia/ml) was inoculated into the stem bases of 10 healthy drunken horse grass seedlings (around 3 weeks old) using a sterile syringe, then wrapped with moistened sterile gauze, while the other 10 drunken horse grass seedlings were injected with sterile water as a control. All seedlings were placed in a greenhouse with a plastic cover at 15-22°C and 90-100% relative humidity. All inoculated drunken horse grass seedlings showed symptoms similar to those of natural infection with stem basal rot, whereas uninoculated drunken horse grass seedlings remained healthy after 15 days. Fungi re-isolated from the basal stems of inoculated plants were confirmed phenotypically and molecularly as F. pseudograminearum. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of drunken horse grass in China. The disease has become a potential threat to the growth of drunken horse grass in China.

First Report of Uromyces aecidiiformis Causing Rust Disease on Fritillaria unibracteata in China.

Fritillaria unibracteata Hsiao et K. C. Hsia is a recognized source of 'Chuanbeimu' in the 'Chinese Pharmacopoeia'. In China, its bulbs have been used as a traditional herbal cough remedy for about 2,000 years. Surveys for fungal diseases were conducted in Xiaojin and Songpan, Sichuan Province, the primary cultivation region of F. unibracteata, with an area of 150 acres, in May and July 2022. Rust was found in almost all areas and incidence ranged from 5% to 80% in all study areas. Diseased leaves displayed yellow spots on the upper side, and raised buff, golden, or fuscous waxy pustules on the lower side. In severe cases, the infection extended to the stems and petioles, leading to wilting and death of plant. Spermogonia, aecia, and telia were mainly found on the underside of leaves. Spermogonia were scattered among the aecia and exhibited a range of colors from honey-yellow to chestnut-brown. They had a cross-sectional diameter of 94.4 to 214.3 µm height and 94.2 to 197.5 µm in width (n=30). They were nearly spherical, embedded in the host tissue, and had distinct periphysis at the pores. Aecia were hemispherical, initially white, with the peridium later turning yellowish-brown and opening via a central pore. Aeciospores were pale yellow, finely and closely verrucose, measuring 20.6 to 34.1 × 18.4 to 30.1 µm with a cell wall thickness of 1.5 to 2.4 µm (n=51). Prior to plants wilting, elongated telia were observed, gradually exposed, then finally opening through longitudinal cracks in the epidermis. Teliospores were unicellular, dark brown, oblong to oval, and solitary on stems, measuring 24.7 to 38.2 × 19.2 to 27.8 µm (n=130) with a wall thickness of 1.6 to 3.1 µm, with a low hyaline papilla at the apex and were moderately rugose with longitudinal parallel ridges. The characteristics align with previous descriptions of Uromyces aecidiiformi (Rees, 1917, Zhuang, 2005). The primer pair LR0R (Moncalvo et al., 1995)/LR5 (Vilgalys & Hester, 1990) was utilized for amplifying and sequencing the large subunit of the nuclear ribosomal RNA genes from strains IS909-3 and IS1816 (GenBank PQ008482, PQ008483). The obtained sequences showed a high similarity of 99.9% to 100% similarity to strains U1023 and UBC19 of U. aecidiiformis in RustHubb (KR0014142 and PUN23000)( Kaishian et al., 2024). Through examination of morphology, host range, and sequence similarity, we determined the rust species to be U. aecidiiformis. Pathogenicity testing was conducted by spraying a suspension of aeciospores (1×105 spores/mL in 0.05% Tween 20 solution) on six healthy four-year-old F. unibracteata plants indoors in May 2023. The plants were allowed to grow under natural conditions, where the diurnal temperature ranged from 9 to 20℃, with an average temperature of 14℃, which is conducive to the growth of F. unibracteata. Another six seedlings were sprayed with 0.05% Tween 20 solution as controls. After three weeks, all infected plants showed symptoms similar to those seen in the field, while control plants remained symptom-free. Microscopic examination and sequencing confirmed that the pathogen morphology was consistent between the field and the inoculation, meeting Koch's postulates. Although U. aecidiiformis has been previously reported to cause rust of F. pallidiflora and F. ussuriensis(Zhuang, 1989, Zhuang, 2005), this is the first report of U. aecidiiformis causing rust on F. unibracteata in China. This pathogen significantly reduces the yield and quality of Chuanbeimu, highlighting the importance of effectively identifying and controlling it.