Fusarium crown rot (FCR) on wheat is a soil-borne disease that affects the yield and quality of the produce. In 2020, 297 Fusarium pseudograminearum isolates were isolated from diseased FCR wheat samples from eight regional areas across Hebei Province in China. Baseline sensitivity of F. pseudograminearum to fludioxonil (0.0613 ± 0.0347 μg/mL) and tebuconazole (0.2328 ± 0.0840 μg/mL) were constructed based on the in vitro tests of 71 and 83 isolates, respectively. The resistance index analysis showed no resistance isolate to fludioxonil but two low-resistance isolates to tebuconazole in 2020. There was an increased frequency of resistant isolates from 2021 to 2022 based on the baseline sensitivity for tebuconazole. There was no cross-resistance between fludioxonil and tebuconazole. This study provides a significant theoretical and practical basis for monitoring the resistance of F. pseudograminearum to fungicides, especially the control of FCR.

China is the largest chili pepper producing country, and Hebei Province stands out as the forth with planting area at about 1500 km2 in China. Pepper (Capsicum annuum L.) is susceptible to Colletotrichum spp. infection during its growth, which seriously affects production yield and quality. In September 2020, widespread anthracnose was observed on pepper in Hebei (115.48° N, 38.77° E), China. Necrotic lesions on pepper fruits were suborbcular, sunken, with acervuli arranged in the middle of lesion (e-Xtra 1A). To perform fungal isolation, small tissue with 0.3 cm2 in size at the symptomatic tissue margin was surface disinfested with 75% ethanol for 10 s, and 0.1% HgCl2 for 40 s, then washed three times with sterile ddH2O. Fragments were placed on potato dextrose agar (PDA) amended with 100 mg·L-1 chloramphenicol and incubated at 28 ºC under darkness for 4 days. One of the strains of Colletotrichum spp., named HQY157, was purified by single-spore isolation, then used for morphological characterization, phylogenetic analysis, and pathogenicity tests. Colonies presented light grey aerial mycelium, occasionally mixed with gray-black strips, and the reverse was similar to the surface on PDA (e-Xtra 1B). Conidia were smooth-walled, aseptate, straight with obtuse to slightly rounded ends, 17.3-28.5 × 3.1-7.4 μm (n=50) (e-Xtra 1C). For molecular identification, the internal transcribed spacer (ITS) region, partial sequences of actin (ACT), β-tublin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS) were sequenced using the specific primers (Weir et al. 2012). Sequences were deposited in GenBank with the following accession numbers OM317600-OM317604. A Maximum-Likelihood phylogenetic tree was constructed, based on the concatenated sequences (ACT, CHS, GAPDH, TUB, and ITS) of HQY157 and other closely matching Colletotrichum species obtained from GenBank, by using MEGA-X. It showed that HQY157 was grouped with the C. sojae with bootstrap values of 100% (e-Xtra 2). To confirm the pathogenicity, surface-sterilized healthy pepper fruits and healthy fruits with wounds (deal with a sterile toothpick after surface-sterilized) were then inoculated with 2 μL of conidial suspension (106 conidia/mL). The fruits inoculated with 2 μL sterile distilled water were taken as negative controls. After inoculation, the fruits were kept in a plastic box with sterilized filter paper moistened with sterilized water, and maintained at 25°C in the dark. The experiment was repeated three times. Anthracnose symptoms were observed 7 days after inoculation on the wounded pepper fruits, whereas the unwounded and negative control fruits remained symptomless (e-Xtra 1D). Colletotrichum sojae was re-isolated from the infected pepper fruits and identified by morphological and molecular analysis, fulfilling Koch's postulates. Colletotrichum sojae occurs mainly on Fabaceae plants such as Glycine max, Medicago sativa, Phaseolus vulgaris, and Vigna unguiculata (Damm et al. 2019, Talhinhas and Baroncelli, 2021), and Panax quinquefolium (Guan et al. 2021). To our knowledge, this is the first report of C. sojae causing anthracnose on pepper in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this chili pepper disease.

Fusarium crown rot (FCR) is an important disease on wheat (Triticum aestivum L.) all over the world. Fusarium pseudograminearum is reported the main causal agent of FCR in China (Deng et al. 2020). In 2020, FCR occurred in wheat in Langfang, Hebei Province (116.31°E, 38.82°N) with observed incidence of 37.2% (48 out of 129 plants in total). The diseased wheat showed brown lesions at the crown and then stem necrosis. Samples with diseased symptom were collected from fields in late May 2020 (at the premature stage, 36 weeks after planting) (e-Xtra 1A). To perform fungal isolation, 0.3 cm2 samples excised at the symptomatic crown were surface disinfested with 75% ethanol for 10 s, and 0.1% HgCl2 for 40 s, then washed three times with sterile ddH2O. When cultured on potato dextrose agar (PDA), the colonies of two isolates out of Langfang (11.8% frequency) initially arewhite becoming violet with age, with violet pigments produced on PDA (e-Xtra 1B). Single-spored isolates were acquired, macroconidia were slender, thin walled, with 3- to 5-septate, measurements of 15.7-31.4 µm × 2.7-6.3 µm (n=50) (e-Xtra 1C). The pure culture were named as HWA94 and HWA97, respectively. DNA was extracted from the single-spored mycelium of HWA97 using the CTAB method (Leslie and Summerell, 2006). Internal transcribed spacer (ITS) region, partial sequences of actin (ACT), translation elongation factor 1-α (EF-1α) gene, 28S ribosomal RNA (LSU), and DNA-dependent RNA polymerase II largest subunit rpb1 (RPB1) were amplified using primers ITS1/ITS4, EF-1F/EF-1R, ACT512F/ACT728R, LR/LROR and RPB1B-F/ RPB1B-R and sequenced. The ITS, EF-1α, ACT, LSU, and RPB1 sequences were deposited in GenBank under accession numbers OM459813 to OM459817. These sequences showed 99.64%, 100%, 100%, 100%, and 100% similarity with the reference strain F. nygamai CBS749.97, respectively, resulting in HWA97 being identified as F. nygamai. To confirm the pathogenicity, inoculum was prepared by inoculating fully colonized F. nygamai (HWA97) PDA plugs on sterile wheat grain medium, cultured 7 days at 25℃ till massive mycelium formed, and hand shaken every two days to mix the wheat grains and the F. nygamai mycelium completely. Ten wheat seeds (cv. Jimai22, susceptible to FCR) for each 10-cm pot were inoculated with 10 g of inoculum when planting, then covered with soil. Mock inoculated wheat seeds with sterile grain without inoculum were used as control. The experiment was conducted in greenhouse, and repeated three times. Symptoms (brown necrosis at the crown) appeared 35 days after inoculation (dai) (e-Xtra 1D), with 91.5% incidence and 49.5±2.6 disease index. Mock-inoculated plants remained symptomless (e-Xtra 1E). Fusarium nygamai was re-isolated from the symptomatic stem and identified by morphological and molecular analysis, fulfilling Koch's postulates. Fusarium nygamai has been previously reported and recovered from wheat root and stalk (Fard et al. 2017) and causes root rot on wheat in Iraq (Minati, 2020), rice in Sardinia (Balmas et al. 2000), sugar beet in China (Cao et al. 2018), as well as lentil (Lens culinaris Medikus) in Pakistan (Rauf et al. 2016). To our knowledge, this is the first report of F. nygamai causing FCR of wheat in China. This study contributes useful information for epidemiologic studies for FCR. Additional studies will be needed to determine the distribution, aggressiveness, and impact on yield of F. nygamai compared with the dominant causal agent F. pseudograminearum.