RESUMO
Paeonia lactiflora Pall is a traditional famous flower with long cultivated history in China, and has important medical and ornamental functions (Duan et al. 2022). In the middle of June 2022, anthracnose disease was observed nearly 25% (n=90) on P. lactiflora in Poyang County, Shangrao City, Jiangxi Province (29.00° N, 116.67° E) (Figure 1 E). The symptoms of the disease were small, round, light brown spots then grew bigger to round or irregular dark brown lesions (5 to 7 mm diameter) progressively on the leaves with disease spread (Figure 1 A). Subsequently, necrotic tissue was formed in the center and caused fade and wilt on the leaves ultimately, which reduced the medicinal and aesthetic value severely. Small pieces of diseased tissue (5 × 5 mm) were cut from the diseased junction, disinfected with 75% ethanol for 30 to 45 seconds, then 1% NaClO for 1 to 2 minutes, rinsed three times with sterile water. To identify the pathogen, tissues were placed on PDA and incubated for 3 days at 28°C. Single spore isolates were cultured on PDA, the colonies of one representative strain (SY4) were originally white with a lot of aerial mycelium after 5 to 7 days at 28°C in the incubator. The center of the colony turned greyish-white, released tiny orange-yellow particles (conidia) (Figure 1 F and 1 G), which were single, colorless, elongated ovals with rounded ends and measured 11.29 to 23.24 × 3.94 to 5.60 µm (av=15.89 µm × 4.74 µm, n=50) (Figure 1 H and 1 I). The isolate SY4 was identified to Colletotrichum fructicola based on morphological characteristics (Yang et al. 2021; Li et al. 2022b). For further molecular identification, the rDNA-ITS, actin gene (ACT), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), chitin synthase (CHS) and calmodulin gene (CAL) genes were amplified and sequenced with primers of ITS1/ITS4 (Gardes et al. 1993), ACT-512F/ACT-783R, GDF/GDR (Templeton et al. 1992), CHS-79F/CHS-345R (Carbone et al. 1999) and CL1C/ CL2C (Weir et al. 2012) respectively. The accession numbers in GenBank were OP523977 (ITS-rDNA), OP547618 (ACT), OP605733 (GAPDH), OP605732 (CHS), and OP605731 (CAL). The BLAST analysis revealed that these sequences were identical more than 99% with those of C. fructicola (GenBank accession Nos. MZ437948.1, MN525803.1, MN525860.1, MZ13360.1 and ON188684.1) (Figure 2). To confirm pathogenicity, the leaves were cleaned with 75% ethanol, rinsed with sterile water. After the leaf surface was dried naturally, 20 leaves were pricked at two symmetrical places on either side of the main veins of the leaf with a sterilized inoculum needle (2.0 mm in diameter), half of the wounded leaves were inoculated with 20 µL spore suspension (1.0 × 106 spores/mL) (Figure 1 C and 1 D), while the other half were inoculated with sterile water as controls (Figure 1 B). Inoculated leaves were grown for 5 days in an incubator at 28 °C and above 90% relative humidity, repeated three times. The results demonstrated that the wounded leaves with C. fructicola showed the same signs of wilting with the original disease leaves, while control leaves remained healthy. The same fungus was reisolated from the diseased leaves which confirmed with Koch's postulates. The same fungus was re-isolated from the diseased leaves while it was not isolated from control leaves, confirmed with Koch's postulates. In China, it had been reported that C. fructicola caused anthracnose on Persea americana (Li et al. 2022a) and Myrica rubra (Li et al. 2022b). To the best of our knowledge, this is the first report of anthracnose on P. lactiflora caused by C. fructicola in China. The results will help to develop effective control strategies for anthracnose on P. lactiflora.
RESUMO
Ponkan (Citrus reticulata Blanco cv. Ponkan) is a Chinese citrus species with tasty fruit. In November 2021, an unknown postharvest disease of Ponkan fruit caused nearly 15% losses of 2000 fruits in Nanchang, Jiangxi Province (28.68° N, 115.85° E). The initial fruit's surface necrosis was brown (Xu et al. 2022) (Figure 1A). Disease spots spread to the entire fruit, white or grey hyphae appeared, and the fruit rotted. Twenty diseased fruits were surface-disinfested with 2% sodium hypochlorite and 75% ethanol, then rinsed with sterile distilled water to isolate the pathogen. Diseased tissue sections (5 × 3 mm) were incubated on potato dextrose agar (PDA) for 7 days at 25°C. Twelve of 15 monoconidial isolates have similar morphology. On PDA, the isolates produced copious white aerial mycelia. After 5-7 days on straw juice medium, two types of conidia appeared (Rice straw 60 g, Agar 20 g, distilled water 1000 mL) (Figure 1E-I). Macroconidia were abundant, falcate, slender, and slightly curved with 0-8 septa, mostly 4-5 septa (average 41.70 × 3.81 m, n=100) (Figure 1J). Microconidia were globose, oval, or piriform with 0-1 septa, 2.72 to 8.57 × 2.53 to 7.47 m (average 5.49 × 4.52 m, n=50) (Figure 1L), and chlamydospores were not observed. Conidial and colony morphology identified 12 monoconidial isolates as Fusarium graminearum (Fisher et al., 1982; Yulfo-Soto et al., 2021). Genomic DNA was extracted from three isolates using a DNA Extraction Kit (Yeasen, Shanghai, China). The ITS1/4 region combined with partial gene fragments of translation elongation factor-1 alpha (TEF-1α, primer TEF1/2, O'Donnell et al. 1998), RNA polymerase second largest subunit (RPB2, primer fRPB2-5F/7cR, Liu et al. 1999) and ß-tubulin (ß-tub, primer Bt2a/2b, Li et al. 2013) from the isolates were amplified and sequenced. The three tested isolates showed identical gene sequences. Sequences amplified from one representative isolate (PG16) have been submitted to GenBank. BLAST searches revealed that ITS (OM019317), TEF-1α (OM048103), RPB2 (ON364348), and ß-tub (OM048104) had 99 to 100% identity compared with F. graminearum (MH591453.1, KX087136.1, MF662636.1, and MZ078952.1, respectively) in GenBank. The phylogenetic analysis combined ITS - TEF-1α - RPB2 (O'Donnell et al. 2015) concatenated sequences using MEGA7.0 (Mao et al. 2021) showed the isolate was clustered with the F. graminearum clade with 100% bootstrap support (Figure 2). The isolate PG16 was used for pathogenicity tests. Ponkan fruits were surface-disinfested with 75% ethanol and rinsed with sterile distilled water three times. Then, 30 punctured wound fruits (2-mm-diameter, 2-mm-depth) with a sterile needle and 30 unwounded fruits were inoculated with conidial suspension (10 µL, 3.0 × 105 conidia/mL). while the control fruits were inoculated with 10 µL sterile distilled water. All fruits were incubated at 25°C and 90% relative humidity. Two days later, all wounded fruits inoculated with conidial suspension showed disease spots, similar symptoms to the original rotten fruits (Figure 1D). Control and conidial-inoculated unwounded fruits were healthy (Figure 1B-C). The Pathogenicity test was repeated twice, and similar symptoms were observed. Morphologically and molecularly, the re-isolated fungus matched the inoculated isolate. First report of F. graminearum causing Ponkan fruit rot in China. As Ponkan is an important citrus crop with high economic value in China, identification of the causing agent, F. graminearum, for fruit rot allows the development of control measures to manage this disease.
RESUMO
Botryosphaeria dothidea is a major postharvest causal agent of soft rot in kiwifruit. Methyl jasmonate (MeJA) is an important plant hormone that participates as a plant defense against pathogens from a signal molecule. However, the impact and regulatory mechanism of MeJA on the attenuation of kiwifruit fungal decay remains unknown. This work investigated the effects of exogenous MeJA on the enzyme activity, metabolite content and gene expression of the phenylpropanoid and jasmonate pathways in kiwifruit. The results revealed that MeJA inhibited the expansion of B. dothidea lesion diameter in kiwifruit (Actinidia chinensis cv. 'Hongyang'), enhanced the activity of enzymes (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate: coenzyme A ligase, cinnamyl alcohol dehydrogenase, peroxidase and polyphenol oxidase), and upregulated the expression of related genes (AcPAL, AcC4H, Ac4CL, and AcCAD). The accumulation of metabolites (total phenolics, flavonoids, chlorogenic acid, caffeic acid and lignin) with inhibitory effects on pathogens was promoted. Moreover, MeJA enhanced the expression of AcLOX, AcAOS, AcAOC, AcOPR3, AcJAR1, AcCOI1 and AcMYC2 and reduced the expression of AcJAZ. These results suggest that MeJA could display a better performance in enhancing the resistance of disease in kiwifruit by regulating the phenylpropanoid pathway and jasmonate pathway.
