Banana (Musa spp.) is a popular fruit all over the world, and it’s also an important cash crop with a planting area of 358,924 ha in southern China. In July 2020, a peduncle soft rot disease occurred on dwarf banana (Musa sp. cv. Guangfen) in Guigang city (N22°50'29″, E109° 43'34″), Guangxi province, China. More than 20% plants were infected in the banana plantation. The first external sign of the disease appeared on the incisional wound after the flower bud was cut off from the peduncle. The symptom initially appeared as a black lesion on the wound, then extended into the internal tissue of the whole peduncle. In the later stages, the internal tissue became soft and rot, occasionally formed a necrotic cavity, and eventually led to the black rot of the whole peduncle with a foul smell. To isolate the pathogen, the internal lesion tissues of 5 mm × 5 mm were collected between the border of symptomatic and healthy tissue, treated with 75% ethanol for 10 s, and 0.1% HgCl2 for 3 min, then rinsed with sterile water for three times. Sterilized tissue fragments were cut to pieces with sterilized surgical shears and soaked in 5 mL sterile water, then shaken for 10 min in a vortex oscillator. The suspension was diluted 1000 times with sterilized water,then plated on nutrient-agar medium and incubated at 28℃ in darkness for 24 h. Among the 32 isolates, 23 pure bacterial cultures with similar morphology were predominantly obtained from the samples. These bacteria were gram-negative, and their colonies were initially yellowish white with irregular edges and smooth surfaces, then turned to grayish blue after 72 h incubated at 28℃. The representative isolates GZF2-2 and GZF1-8 were selected for further identification. Genomic DNA was isolated from the bacteria and the 16S rDNA was amplified with primers 27F/1492R (Weisburg et al. 1991) and sequenced. The obtained sequences (GenBank Accession No. MZ768922 and OK668082) showed >99% identities to several records of Dickeya fangzhongdai deposited in NCBI GenBank (1400/1404 bps for GZF2-2 to KT992690, 1409/1417 bps for GZF1-8 to MT613398) based on BLAST analysis. In addition, the recA, fusA, gapA, purA, rplB, dnaX genes and the 16S-23S intergenic spacer (IGS) regions of the two isolates were also amplified and sequenced (GenBank Accession Nos. OK634381-OK634382, OK634369- OK634370, OK634373-OK634374, OK634377-OK634378, OK634385-OK634386, OK634365- OK634366 and OK631722-OK631723) as described by Tian et al. (2016). All the DNA sequences matched that of D. fangzhongdai strains JS5T (percent identities>99.06%), PA1 and ECM-1 in GenBank. Neighbor-joining phylogenetic analysis by software MegaX (Kumar et al. 2018) based on the 16S rDNA sequences revealed that the two isolates were in the same clade with reported D. fangzhongdai strains. Multilocus sequence analysis of the other seven regions also showed the two representative isolates were belong to D. fangzhongdai. Therefore, the isolates were identified as D. fangzhongdai. Pathogenicity of isolate GZF2-2 was investigated to demonstrate Koch’s postulate. The end of the banana peduncles of 6 healthy plants were cut off, and 10 mL bacterial suspension (108 CFU/mL) was inoculated to the fresh wound on the plants using sterile brushes. Six control plants were inoculated with sterilized water. All the inoculated peduncles were covered with plastic bags to maintain high humidity. After 28 days, all the peduncles inoculated with strain GZF2-2 showed soft rot symptoms similar to those observed in the field, while the controls remained symptomless. The same bacteria were re-isolated from the symptomatic peduncles and confirmed by sequencing the 16S rDNA. D. fangzhongdai has been reported to cause soft rot on onion (Ma et al. 2020) and bleeding cankers on pear trees (Chen et al. 2020). To the best of our knowledge, this is the first report of D. fangzhongdai causing peduncle soft rot on banana in China.
The plant pathogenic fungus, Sclerotinia minor IMI 344141, has been developed as a bioherbicide for broadleaf weed control in turfgrass and a means to differentiate this biocontrol agent from like organisms is required. A strain specific molecular marker was developed to detect and monitor the Sclerotinia minor IMI 344141 bioherbicide strain. The method was based on polymerase chain reaction (PCR) amplification of two sequence-characterized amplified regions (SCAR) primer pairs for a first round PCR, and another two sets of nested primers was used for a second round PCR if higher sensitivity was needed. Sclerotinia minor IMI 344141 was successfully traced from both pure cultures and environmental samples originating from bioherbicide-released field trials. DNA of the S. minor bioherbicide isolate IMI 344141 was detected in the soil 2 months after application, but was not detected in the 3-and 9-month samples after application. When applied as a bioherbicide, S. minor (IMI 344141) did not persist into the following spring season in turf environments. This molecular detection method provides a mechanism to distinguish this isolate from related organisms and a tool to monitor behavior of the biocontrol agent S. minor IMI 344141 in nature, particularly in soil.
Fusarium wilt of banana is a devastating disease caused by Fusarium oxysporum f. sp. cubense (Foc). It has restricted the development of the banana industry worldwide and is particularly serious in China due to the large planting areas and special planting patterns. However, there is no rapid and accurate approach to detect the Foc strains that specifically occur in China due to the rich genetic diversity observed in this pathosystem. In this study, we evaluated the performance of 10 PCR previously published primer pairs on 103 representative Foc strains in China and neighbor countries and screened out a set of primers (Foc-specific primer pair SIX9-Foc-F/R, Foc R1-specific primer pair SIX6b-210-F/R, Foc R4-specific primer pair Foc-1/2, and Foc TR4-specific primer pair W2987F/R) suitable for the detection of Foc strains in China and the surrounding Southeast Asian countries. Moreover, we developed a molecular detection system to accurately identify the different physiological races of Foc. The findings of this study provide technical support for preventing and controlling the spread of Fusarium wilt of banana in the field in China.
Geodorum eulophioides Schltr. is a critically endangered orchid listed in the International Union for Conservation of Nature (IUCN) Red List of threatened species. At present, only two natural populations were found in China. It has important scientific and ornamental values because of its uniqueness. During the summer of 2019, a black leaf spot disease occurred on G. eulophioides, in Yachang Orchid National Nature Reserve (E106°13′32″,N24°44′19″) in Guangxi province, China. More than 60% of leaves of these plants were infected. The disease symptoms initially appeared as small yellow circular spots, which enlarged into irregular brown spots (6 to 9 cm length and 3 to 5 cm width). In later stages of the disease development, the center of the spots became dark brown with a clear edge and surrounded by a yellow halo. In severe infections, the spots coalesced covering the entire leaf. Six symptomatic leaves were collected from three infected plants, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 4 min, and subsequently washed three times with sterile water, then plated onto potato dextrose agar (PDA), and incubated at 28℃ for three days. Eighteen fungal cultures with similar morphological characteristics were obtained from the infected tissues. Colonies were initially white, then turned dark grey after nine days. To induce sporulation, isolates were grown on 2% water agar and incubated under UVA light at 28℃ for nine days. Three isolates were selected for morphological characterization. Conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.7 to 16.6 μm (avg. 13.8 μm) × 4.1 to 6.7 μm (avg. 5.1 μm) (n=50). The isolate DBL-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (EF1-α), and beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R(Alves et al. 2008;Carbone & Kohn, 1999), and T1/T2 (O’Donnell et al., 1997), respectively. The obtained ITS sequence (GenBank Accession No. MN918440), EF1-α sequence (MN963815), and TUB2 sequence (MN963816) showed >99% homology with several GenBank sequences of Neofusicoccum parvum (JX513636, KU997497 for ITS, KU997261, MH252401 for EF1-α, and KJ841779, MK412882 for TUB2, respectively). Based on morphological characteristics of the asexual morph and maximum likelihood analyses of a combined rDNA-ITS, EF1-α and TUB2 gene sequences, was identified as N. parvum. Pathogenicity test was performed using isolate DBL-1 by inoculating 3 leaves of G. eulophioides plants. The test was repeated three times. Each leaf was wounded using a sterile needle, and a mycelial plug (6 mm diameter) harvested from the periphery of a 3-day-old colony grown on PDA was placed on each wound. Plants were then covered with plastic bags to maintain high relative humidity of 90% and kept at 28℃ in a greenhouse under natural daylight conditions. An equal number of leaves on the same plant were inoculated using sterile PDA plugs and served as mock inoculated controls. After three days, all the inoculated leaves showed black spot symptoms resembling those observed in the field, whereas controls remained symptomless. The fungus was re-isolated from the symptomatic leaves, thus completing Koch’s postulates. N. parvum has been reported to cause leaf spot disease on Myristica fragrans (Jayakumar, et al., 2011), Ginkgo biloba (Mirhosseini, et al., 2014), Vitis heyneana (Wu, et al., 2015), and Hevea brasiliensis (Liu et al., 2017), respectively. To the best of our knowledge, this is the first report of N. parvum causing leaf spot disease on G. eulophioides in China. The disease control measures and in-situ conservation method need to be strengthened to protect this rare species.
Pomegranate (Punica granatum L.) is a deciduous shrub or small tree that is native to Iran and Afghanistan. It is also a commercially important fruit tree in China and worldwide. In the summer of 2022, a serious root rot disease occurred in some pomegranate orchards in Xichuan County(32º42´ N, 111º48´ E), Henan Province, China, with an incidence of ~30%. Symptoms included leaf yellowing and wilting, root browning and rotting, and stem-base cracking, eventually leading to defoliation and death. To isolate the causal agent, small pieces (5×5 mm) of diseased root from six trees were surface-sterilized by dipping in 2% NaClO for 8 min followed by 70% ethanol for 15 s, rinsed five times with sterile water, and plated on potato dextrose agar (PDA), then incubated at 28°C in the dark for 5 days. Fifteen pure fungal isolates with the same morphological characteristics were obtained from 24 pieces of roots. All isolates produced white fluffy mycelia. Microconidia were hyaline, oval or reniform, with zero to one septa and dimensions of 7.1 to 19.9 (average 14.5 )× 3.8 to 8.0 (average 5.6) μm (n = 100). Macroconidia were sickle-shaped, one to four septate, and 20.1 to 40.8 (average 26.5) × 4.8 to 8.6 (average 6.5) μm (n = 100). Chlamydospores were spherical, single, in pairs or chains, and 5.6 to 9.8 (average 6.8) µm in diameter (n = 100). Based on the above characteristics, the pathogens were identified as Fusarium sp. (Leslie and Summerell 2006). Genomic DNA was extracted from mycelia of two representative isolates Fs1 and Fs3. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4, EF1/EF2, and RPB2-5f2/RPB2-7cr, RPB2-7cf/RPB2-11ar (O’Donnell et al., 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates Fs1(GenBank accession nos. OK001765, OQ921726 and OQ928396) and Fs3 (GenBank accession nos. OK001771, OQ921727 and OQ928397) showed 99%-100% identity with multiple GenBank sequences of Fusarium falciforme (KY617066, MN064683, KF255514, OQ933361, KY556711 and ON331935). A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analysis revealed that both isolates Fs1 and Fs3 were in the same clade with F. falciforme strains. Based on the morphological and molecular characteristics, the isolates were identified as members of F. falciforme. For pathogenicity testing, conidial suspensions (1×108 spores /mL) of isolates Fs1 and Fs3 were poured onto the roots of healthy pomegranate that had been planted in pots two months previously. Ten plants were inoculated for each isolate. Control plants were drenched with sterile water. After 3 months, inoculated plants developed leaf yellowing and wilting accompanied by root browning and rotting, much like symptoms observed in field plants. The same fungi re-isolated from the experimental plants were confirmed to be F. falciforme by morphology and sequence analysis. This is the first report of F. falciforme causing root rot on pomegranate. F. falciforme is a ubiquitous soil-borne pathogen that causes root rot on multiple plants around the world (Xu F., et al. 2022; Qiu R., et al. 2023). The results of pathogen identification are essential precursors to development of effective control of the disease.
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