Search

In July 2022, dieback and discoloration were detected on infected stems of peanut in Qijiang District of Chongqing (106.56°E,29.41°N), China, with an incidence up to 5%. These peanut stems had disease symptoms typical of anthracnose with irregular gray-brown spots with dark brown edges, sunken, and necrotic. High temperature and high humidity were favorable for the growth of the pathogen. To isolate the pathogen, we collected 10 typical infected peanuts and cut one piece from each of symptomatic stems, surface sterilized with 0.5% NaClO for 1 min, and 75% ethanol for 30 s, then rinsed three times with sterile distilled water and dried on sterilized filter paper. These pieces were incubated on potato dextrose agar (PDA) at 25°C in the dark. Pure cultures were obtained from hyphal tips of each colony. It was found that isolates with the same colony morphology were isolated from each infected stem. A representative isolate (L7) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. The colonies appeared white to gray, with white margins and aerial hyphae, and the reverse of the colonies was gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, 13.4 to 18.8 × 4.2 to 5.8 μm (n=50). Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Cannon et al., 2012). For molecular identification, genomic DNA was extracted using a CTAB method and partial sequences of β-tubulin (TUB2), actin (ACT) genes, chitin synthase (CHS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced using primers T1/T2, ACT-512F/ACT-783R, CHS-79F/CHS-345R, and GDF1/GDR1, respectively (Damm et al., 2012; Dowling et al., 2020). Using the BLAST, TUB2, ACT, CHS and GAPDH gene sequences (GenBank accession No. OR714793, OP168707, OP168708 and OR714794, respectively) were100% (429 bp out of 429 bp), 99.22% (256 bp out of 258 bp), 99.64% (276 bp out of 277 bp) and 100% (253 bp out of 253 bp) identical to C. gloeosporioides CBS:112999 (JQ005587, JQ005500, JQ005326, and JQ005239), respectively. Using Neighbor-Joining algorithm, phylogenetic analysis was conducted based on the concatenated sequences of published TUB2, ACT, CHS and GAPDH genes. The identified isolate (L7) was closely related to C. gloeosporioides. To evaluate pathogenicity, the stems of ten peanut (Zhonghua12) seedlings (2 weeks) were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) or 20 μl of conidial suspension (105/ml) were inoculated. Non-colonized agar plugs or 20 μl of sterile distilled water were treated as control. After inoculation, the peanuts were kept in a moist chamber at 28°C with 80% humidity in the dark for 24 h, and subsequently transferred to the moist chamber with 12 h light and darkness cycle for 6 days, similar symptoms were observed on all inoculated peanuts. Controls remained asymptomatic. C. gloeosporioides was reisolated from the diseased stems and confirmed using morphological features and sequence analysis of TUB2, ACT, CHS and GAPDH. Anthracnose caused by C. truncatum and C. fructicola has been reported on peanut leaves in China (Gong et al., 2023; Yu et al., 2019). To our knowledge, this is the first report of anthracnose on peanut stem caused by C. gloeosporioides in Chongqing. Our report will provide crucial information for studying on epidemiology and management of this disease.

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.

Peanut (Arachis hypogaea L.) is one of the most economically important crops as a major source of edible oil and protein. In July 2021, a root rot disease was observed on peanut in Laiwu (36º22' N, 117º67' E), Shandong Province, China. Disease incidence was approximately 35%. Disease symptoms included root rot, vessels with a brown to dark brown discoloration, plus progressive yellowing and wilting of leaves from the base leading to whole plant death. To determine the causal agent, symptomatic roots with typical lesions were cut into small pieces, surface sterilized in 75% ethanol for 30 s, and 2% NaClO for 5 min, rinsed three times in sterile water and placed on potato dextrose agar (PDA) at 25℃ (Leslie and Summerell 2006). After 3 days of incubation, whitish-pink to red colonies growing from the roots were observed. Eight single-spore isolates had identical morphological traits that were similar to those of Fusarium spp. A representative isolate (LW-5) was used for morphological characterization, molecular analysis, and pathogenicity test. On PDA, the isolate formed dense aerial mycelia, which were initially white, then became deep pink with age and formed red pigments in the medium. On carnation leaf agar (CLA), macroconidia with 3 to 5 septa were abundant, relatively slender, curved to lunate, that measured 23.7 to 52.2 × 3.6 to 5.4 μm (n=50). Microconidia were oval, 0 to 1 septa. Chlamydospores were globose with a smooth outer wall in chains or single. Following DNA extraction of isolate LW-5, primers EF1-728F/EF1-986R (Carbone et al., 1999), RPB1U/RPB1R, and RPB2U/RPB2R (Ponts et al., 2020) were used to amplify the partial translation elongation factor 1 alpha (TEF1-α), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) regions for DNA sequencing, respectively. BLASTn analysis of TEF1-α (GenBank accession No. OP838084), RPB1 (OP838085), and RPB2 (OP838086) sequences, revealed 99.66, 99.87, and 99.09% identity with those of F. acuminatum (OL772800, OL772952 and OL773104), respectively. Isolate LW-5 was identified as F. acuminatum based on morphology and molecular analysis. Twenty Huayu36 peanut seeds were each planted in a 500-ml sterile pot containing 300 g of autoclaved potting medium (nutritive soil: vermiculite=2:1 in volume). Two weeks after seedling emergence, 1 cm depth of the potting medium was dug around the plants to expose the taproot. Two 5-mm wounds per taproot were scratched with a sterile syringe needle. Potting medium in each pot of 10 inoculated plants was mixed with 5 ml of conidial suspension (106 conidia per ml). The other 10 plants were used as non-inoculated controls and treated with sterile water in the same manner. The seedlings were placed in a plant growth chamber maintained at 25°C, RH >70%, 16-h light per day, and irrigated with sterile water. After 4 weeks, inoculated plants exhibited yellowing and wilting symptoms that were similar to those observed in the field, while non-inoculated control plants had no symptoms. F. acuminatum was re-isolated from diseased roots and confirmed using morphological features and DNA sequence analysis of TEF1-α, RPB1 and RPB2. F. acuminatum was reported to cause root rot on Ophiopogon japonicus (Linn. f.) (Tang et al., 2020), Polygonatum odoratum (Li et al., 2021), and Schisandra chinensis (Shen et al., 2022) in China. To our knowledge, this is the first report of root rot on peanut caused by F. acuminatum in Shandong Province, China. Our report will provide crucial information for studying the epidemiology and management of this disease.

In June 2021, a previously unreported leaf blight disease of peanut (Arachis hypogaea) was observed on field-grown peanut (Jinhua19) in Laixi city, Shandong province of China. Approximately 5% of plants showed disease symptoms in the fields we investigated. The symptoms first appeared as yellow round or irregular spots on leaves, and then the spots became brown. As the disease progressed, spots became larger and even converge, which later produced leaf chlorosis and abscission. Symptomatic leaves were cut into small pieces, surface disinfested with 70% ethanol for 30s, 1% NaClO for 60s, rinsed three times in sterile water, dried on sterile filter papers, placed on potato dextrose agar (PDA) media, and incubated at 25°C in darkness. Fungal cultures were initially white, with red pigment, then turned gray, and eventually turned black, and aerial hyphae were dense. Conidia were spherical or slightly ellipsoidal, black, smooth, and 8.6 to 11.5 × 8.7 to 14.5μm (n=50). Morphological characteristics of the isolates matched the description of Nigrospora aurantiaca (Wang et al. 2017). Molecular identification was performed by sequencing beta tubulin gene (TUB) with Bt2a/Bt2b and translation elongation factor 1-alpha (TEF) with EF1-728F/EF1-986R (Wang et al. 2021) of a representative isolate ZHX11. TUB (OK489789) and TEF (OK489790) of ZHX11 obtained 100% (401/401 nucleotides) and 99.64% (279/279 nucleotides) similar to those of N. aurantiaca (MN329935, MN264010), respectively. Alignment was conducted separately for each gene set using Clustal W algorithm implemented in MEGA 7.0 (Kumar et al. 2016), and multi-gene (TUB and TEF) phylogenetic analyses using Neighbor-Joining (NJ) method showed that the isolate was N. aurantiaca. To complete Koch's postulates, nine 2-week-old peanut (Zhonghua 12) seedlings were sprayed with conidia suspensions (106 conidia mL-1 in 0.05% Tween 20 buffer). The same number of seedlings were only treated with 0.05% Tween buffer as controls. The experiment was repeated three times. Plants were incubated in a growth chamber (30°C in the day and 25°C at night, a 12-h photoperiod and 80% RH). Ten days after inoculation, typical symptoms were observed on inoculated leaves but not on the controls. N. aurantiaca was reisolated from the diseased leaves but not from the controls. N. sphaerica was observed on peanut in China (Liu et al. 2020). To our knowledge, this is the first report of N. aurantiaca causing leaf blight on peanut in shandong province, China. These findings will help to develop better preventive measures in accordance with the emergence of the new disease.

Peanut (Arachis hypogaea L.) is one of the most economically important crops as an important source of edible oil and protein. In August 2020, circular to oval-shaped brown leaf spots (2-6 mm in diameter) with well-defined borders surrounded by a yellow margin were observed on peanut plant leaves in Laixi City, Shandong Province, China. Symptomatic plants randomly distributed in the field, the incidence was approximately 5%. Leave samples were collected consisted of diseased tissue and the adjacent healthy tissue. The samples were dipped in a 70% (v/v) ethanol solution for 30 s and then soaked in a 0.1% (w/v) mercuric chloride solution for 60 s. The surface-sterilized tissues were then rinsed three times with sterile distilled water, dried and placed on Czapek Dox agar supplemented with 100 μg/ml of chloramphenicol. The cultures were incubated in darkness at 25 °C for 3-5 days. Fungal colonies were initially white and radial, turning to orange-brown in color, with abundant aerial mycelia. Macroconidia were abundant, 4 to 7 septate, with a dorsiventral curvature, and were 3.3-4.5 × 18.5-38.1 μm (n=100) in size; microconidia were absent; chlamydospores were produced in chains or clumps, ellipsoidal to subglobose, and thick walled. The morphological characteristics of the conidia were consistent with those of Fusarium spp. To identify the fungus, an EasyPure Genomic DNA Kit (TransGEN, Beijing, China) was used to extract the total genomic DNA from mycelia. The internal transcribed spacer region (ITS rDNA) and the translation elongation factor 1-α gene (TEF1) were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O'Donnell et al. 1998), respectively. Based on BLAST analysis, sequences of ITS (MT928727) and TEF1 (MT952337) showed 99.64% and 100% similarity to the ITS (MT939248.1), TEF1 (GQ505636.1) of F. ipomoeae isolates. Sequence analysis confirmed that the fungus isolated from the infected peanut was F. ipomoeae (Xia et al. 2019). The pathogenicity of the fungus was tested in the greenhouse. Twenty two-week-old peanut seedlings (cv. Huayu20) grown in 20-cm pots (containing autoclaved soil) were sprayed with a conidial suspension (105 ml-1) from a 15-day-old culture. Control plants were sprayed with distilled water. The experiment was conducted as a randomized complete block design, and placed at 25 °C under a 12-h photoperiod with 90% humidity. Symptoms similar to those in the field were observed on leaves treated with the conidial suspension ten days after inoculation, but not on control plants. F. ipomoeae was re-isolated from symptomatic leaves but not from the control plants. Reisolation of F. ipomoeae from inoculated plants fulfilled Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing peanut leaf spot in China. Our report indicates the potential spread of this pathogen in China and a systematic survey is required to develop effective disease management strategies.