Occurrence of Macrophomina phaseolina Causing Root and Crown Rot on Alfalfa (Medicago sativa) in China

Abstract

Alfalfa (Medicago sativa) is the widest grown and most important sown pasture crop in China and worldwide. Root and crown rot disease severely threatens alfalfa production, causing estimated annual yield losses of up to 20 to 40% worldwide (Fang et al. 2019). Field surveys in major alfalfa-growing areas in northwest China during summer (June to August) in 2017 to 2019 showed root and crown rot was common in fields established for more than 2 years with disease incidence ranging from 30 to 80%. Diseased plants were usually stunted and wilted; taproots and crowns exhibited red-brown to dark-brown discoloration in the cortex and/or vascular tissues. Frequently, lateral roots and stem tissues showed similar symptoms. Whole roots and crowns were decayed, with shoots wilted in severely affected plants. A total of 250 symptomatic plants were sampled randomly in Gansu Province from five alfalfa fields surveyed in 2018. Isolations were conducted by plating discolored root and crown tissues (40 pieces for each plant) sterilized with 1.25% sodium hypochlorite onto water agar. The plates were incubated at 22°C for 3 days, and then growing hyphae were subcultured onto potato dextrose agar (PDA). Twenty-one isolates from the fields in Zhangye, Gansu Province, were primarily identified as Macrophomina phaseolina based on cultural and morphological characteristics on PDA (Kaur et al. 2012; Sanchez et al. 2013). Five isolates picked randomly were purified by single-sclerotial isolation and maintained on 1/5 strength PDA at 4°C for further study. DNA of these isolates was extracted (Cenis 1992), and PCR amplifications of the internal transcribed spacer (ITS) region of rDNA (ITS1, 5.8S rDNA, and ITS2) were performed using primers ITS1 and ITS4. Sequences were then compared with the reference sequences in GenBank using BLAST search, showing sequence identity with M. phaseolina and the best matches at 98.47% (e.g., accession nos. MK408587 and KR012878). The ITS sequence of a representative isolate (Mp3) has been deposited in GenBank (accession no. MT192442). Pathogenicity tests of two isolates (Mp3 and Mp5) were conducted on 2-week-old alfalfa seedlings (cv. Longdong) with three to four leaves grown from surface-sterilized seeds. Seedlings were transplanted into pots (12 × 12 cm) containing pasteurized potting mix infested with mycelial-colonized millet seed inoculum of either isolate at a rate of 0.5% (w/w). Control seedlings were similarly transplanted into pots but with uninfested potting mix. Four seedlings were planted in a pot with 12 replicate pots for each treatment, and pots were watered every 2 days to free draining under controlled environment conditions (28°C, 12-h photoperiod, and 65% relative humidity). Three weeks postinoculation, all inoculated plants showed stunting symptoms, with discolored roots and crowns with a disease incidence of 100%, whereas the control plants showed no disease symptoms. To confirm Koch’s postulates, both isolates were reisolated from discolored root and crown tissues and again confirmed by morphological similarities and ITS sequencing as the inoculated isolates. Five purified isolates have been submitted to the Grassland Culture Collection Center, Lanzhou University (accession nos. LZU-MsR-Mp1 to Mp5). To our knowledge, this is the first report of M. phaseolina causing root and crown rot on alfalfa in China. M. phaseolina has been reported to cause root and crown rot on alfalfa in America, and on other legumes such as white clover in America, mungbean in India, and many other important crops worldwide (Kaur et al. 2012; Pandey et al. 2020; Pratt et al. 1998). Due to its broad host range and potential to be a significant pathogen in alfalfa-growing areas in China, further study on disease epidemic development is recommended.