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Fusarium solani causing fruit rot of peach (Prunus persica) in Hunan, China
Crop Protection 122 (2019) 171–174
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Short communication
Fusarium solani causing fruit rot of peach (Prunus persica) in Hunan, China Jun Zi Zhu 1, Chang Xin Li 1, Chao Jun Zhang, Ying Wang, Xiao Gang Li **, Jie Zhong * Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests, Hunan Agricultural University, Nongda Road 1, Furong District, Changsha City, Hunan Province, 410128, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Fusarium solani Prunus persica Fruit rot disease Identification
During 2017 to 2018, a fruit rot of peach (Prunus persica) occurred in Hunan Province of China. The symptoms showed as soft, brown water-soaked lesions with offwhite sporulation, in some cases, on the fruit surface, extending in the flesh, which was decayed. The fungus was isolated and identified as Fusarium solani based on morphological characteristics and molecular identification using the internal transcribed spacer (ITS), a portion of the translation elongation factor-1 alpha (EF-1α) and beta-tubulin gene sequences. The pathogenicity of the isolated F. solani was confirmed. To our knowledge, this is the first report of F. solani causing fruit rot of peach in Hunan, China.
1. Introduction
2. Material and methods
Peach [Prunus persica (L.) Batsch], belonging to the Prunus genus in family Rosaceae, is the third most important deciduous fruit tree in the world. China is the country with the largest peach cultivation area and yield (Verde et al., 2013). Many fungal diseases are commonly wide spread during production, storage and marketing of this fruit. Disease control is vital for the production of peach fruits. Fusarium solani is a species complex (FSSC) frequently isolated from soils, plants, animals, insects and humans that act as decomposers, endophyte, nutritional symbiont and pathogens (Booth, 1971; Schroers et al., 2016). This species represents a phylogenetically and biologically complex group accommodating several formae speciales and biological species. Recently, many molecular identification strategies, such as sequencing of internal transcribed spacer (ITS) regions and translation elongation factor-1α (TEF-1α) have been developed for identification of this species (Nalim et al., 2011; Chehri et al., 2015). During August in 2017 and 2018, fruit rot symptoms were observed in a peach orchard (Cuimitao) in the Hunan Province of China (25.73� N, 112.73� E), with about 50% of fruits infected. The symptoms showed as soft, brown water-soaked lesions with offwhite sporulation, in some cases, on the fruit surface, extending into the flesh, which was decayed. This study was conducted to determine the causal agent of this fruit rot disease of peach via morphological observation, molecular identifica tion and pathogenicity tests.
2.1. Sampling and pathogen isolation During 2017 to 2018, samples of infected fruits displaying soft, brown water-soaked lesions were arbitrarily collected from a peach orchard, and 15 fruit samples from 8 different peach trees were used for fungal isolation. Symptomatic tissues were excised at the border of le sions, surface disinfected with 70% ethanol for 30s, 0.1% HgCl2 for 1.5min, rinsed in sterile distilled water, and then plated onto potato dextrose agar (PDA, amended with 100 mg L 1 streptomycin sulphate) and incubated at 26 � C in the dark. After 2–3 days of incubation, mycelia plugs grown from the infected tissues were transferred to fresh PDA for subculture. Pure cultures were obtained by single conidial isolation. 2.2. Morphological and molecular characteristics A total of 15 isolates were recovered. Since all recovered isolates were identical in colony morphology on PDA, one isolate, TZ4, was used as a representative for further evaluation in morphological and cultural characterization. Mycelium plugs (5 mm in diameter) aseptically picked from the edge of actively growing cultures were transferred on PDA and carnation leaf agar (CLA). The morphology and color of the cultures were examined, and the colony growth rate was assessed in millimeter per day by measuring colony diameters daily for at least 7 days. When
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X.G. Li), [email protected] (J. Zhong). 1 Jun Zi Zhu and Chang Xin Li contributed equally to this work. https://doi.org/10.1016/j.cropro.2019.05.009 Received 11 December 2018; Received in revised form 2 May 2019; Accepted 10 May 2019 Available online 11 May 2019 0261-2194/© 2019 Published by Elsevier Ltd.
J.Z. Zhu et al.
Crop Protection 122 (2019) 171–174
master mix (containing dNTPs), 1 μl (10 μM) of each primer set, 1 μl of genomic DNA (10 ng), and 12 μl of sterile double distilled water. PCR conditions included an initial denaturation at 94 � C for 5 min, followed by 35 cycles of denaturation at 94 � C for 30 s, annealing at 55–57 � C for 1 min, extension at 72 � C for 3 min, and a final extension at 72 � C for 10 min. The PCR products were verified by 1% agarose gel electropho resis and sequenced using Sanger sequencing. Generated sequences were analyzed by Blastn against to publicly available sequences in National Center for Biotechnology Information. Nucleotide sequences were aligned using CLUSTALW implemented in MEGA7 (Kumar et al., 2016) and phylogenetic tree was constructed using neighbour-joining method, with a bootstrap test of 1000 replications. 2.3. Pathogenicity tests Pathogenicity was determined on surface-sterilized matured peach fruits. Mycelial plugs (5 mm in diameter) were taken from a colony actively growing on PDA for five days. The fruits were surfacedisinfected with 75% ethanol for 5 s and then inoculated with myce lial plugs. Sterile agar plugs without mycelial were used as control. All the inoculations were maintained at humid chambers at 26 � C. The tests were repeated twice, with each one having five peach fruits. Symptoms were observed four days after inoculation. Fungi were re-isolated and reidentified from infected fruits showing symptoms to verify Koch’s postulates.
Fig. 1. Symptoms of fruit rot on peach fruits (a) Rot symptom was observed on peach fruits in an orchard; (b) pathogenicity test on wounded peach fruit with mycelial plugs (inoculated) showing symptoms 4 and 10 days post-inoculation; check (CK) remained symptomless.
3. Results and discussion Fruit rot symptoms were observed in a peach orchard in the Hunan Province of China. Soft, water-soaked lesions occurred on the outer surface of the fruit, and then extended to the whole fruit with inner tissues becoming brown and decayed (Fig. 1a). Decayed symptoms were also observed in the pedicle often leading to fruit dropping. From 15 symptomatic fruits collected in the field, fungal strains with similar colony characteristics were consistently isolated and cultured on PDA. Colonies showed a brown pigment in the media center, and sparse or white aerial hypha, with a growth rate of 0.46 cm/day on PDA. In addition, we cultured the fungus on PSA and SNA (Spezieller N€ ahrstoffarmer agar) for morphological identification. As expected, the fungus showed distinct culture morphology when cultured on different mediums, showing lesser pigmentation on PAS medium and thinner hyphae on SNA medium (Fig. 2a). When cultured on Carnation leaf agar (CLA) for 10 days at 26 � C, macroconidia were abundantly produced, cylindrical in shape, 30.4 to 41.3 � 4.4–5.4 μm in size, and with 3–5 transverse septa (n ¼ 50); microconidia were also observed hyaline, oval or fusiform, 6.7 to 15.6 � 3.1–4.7 μm and 0 to 3 transverse septa (n ¼ 50) (Fig. 2b). These morphological and cultural characteristics were consistent with de scriptions of F. solani (Booth, 1971). The pathogen was further confirmed by molecular identification. The gene regions of ITS, EF-1α and beta-tubulin were PCR amplified and sequenced. These sequences were deposited in GenBank (GenBank Accession Nos. MG203887, MG205523 and MG205524 for ITS, EF-1α and beta-tubulin, respectively). BLASTn homology search showed that the ITS, EF-1α and beta-tubulin showed 99% identities to the corre sponding sequences of F. solani strains whose GenBank Accession Nos were available (KF030977.1, DQ247569.1 and KJ720638.1, respec tively) (da Silva-Rocha., 2015; Zhang et al., 2006; Su et al., 2014). Phylogenetic analysis were performed based on multiple alignments of the sequenced ITS, EF-1α and beta-tubulin with other corresponding sequences of Fusarium spp. In each of the three phylogenetic trees, the representative strain TZ4 obtained in this work was clustered with F. solani strains (Fig. 3). Based on the morphological characteristics and molecular identification, this fungus was tentatively identified as F. solani (Mart.) L. Lombard & Crous (Leslie et al., 2006; Lombard et al., 2015).
Fig. 2. Cultural and morphology of Fusarium solani isolate in this study. (a) Colony morphology of the representative isolate, TZ4, cultured on PDA, PSA, and NSA, respectively; (b) Microscopic observation of the macroconidia and microconidia of the F. solani isolate.
cultured on CLA for 10 days at 26 � C, conidia dispersed in water were visualized by a light microscope at 400�magnification. The length and width of at least 50 conidia were measured. For molecular identification, genomic DNA was extracted from mycelium by CTAB method as described previously (White et al., 1990). Internal transcribed spacer (ITS) region of rDNA, a portion of the translation elongation factor-1 alpha (EF-1α) gene and beta-tubulin gene were used as target genes to identify the causal fungal organism (White et al., 1990; Carbone et al., 1999; Glass et al., 1995). Target genes were amplified using specific primer pair sets using PCR, where each reaction was a 50 μl reaction containing 25 μl of a PCR 172
J.Z. Zhu et al.
Crop Protection 122 (2019) 171–174
Fig. 3. Phylogenetic analysis of the F. solani isolate TZ4. Phylogenetic trees were inferred from ITS (a), EF-1α (b) and beta-tubulin gene (c) sequences of F. solani and other Fusarium spp. using the neighbour-joining method by MEGA 7.0. Bootstrap values supporting the branch were showed at the nodes. The scale bar indicated the number of nucleotide substitutions per site.
References
Pathogenicity was determined by placing the mycelial plugs on the surface of matured peach fruits. Four days post inoculation, inoculated fruits showed the decayed symptoms similar to naturally infected orig inal fruits, but the control fruits remained symptomless. After 10 days, nearly one third of surface area of the inoculated fruits rotted on which grey white hypha occurred (Fig. 1b). F. solani was specifically re-isolated and confirmed via morphological characteristics and molecular identi fication from inner tissue of the inoculated fruits, confirming Koch’s postulates. China is the leading producer of peaches worldwide. F. solani is a plant pathogen detrimental to many plants including both agricultural crops and trees (Coleman et al., 2016; Farr and Rossman, 2018). For example, it has been reported to cause symptoms of wilt on Melia dubia (Pandey et al., 2018), fruit rot on Eriobotrya japonica (Abbas et al., 2017), root rot on Juglans sigllata Dode (Zheng et al., 2015), soft rot on the tuber of Gastrodia elata (Han et al., 2017), stem canker on Tectona grandis (Huang et al., 2017) and postharvest fruit rot on kiwifruit (Yang et al., 2018). However, until now, F. solani has not been reported as a pathogen of peach. Since this pathogen could be a potential threat on peach production leading to heavy loss in peach in the Hunan province, appropriate chemical and cultural management strategies should be adopted to control spread. Peach fruit rots caused by other Fusarium species have been identified in China, such as F. proliferatun on peach in Ningde, China (Xie et al., 2018). However, to our knowledge this is the first report of F. solani causing fruit rot of peach in Hunan, China.
Abbas, M.F., Naz, F., Rauf, C.A., Mehmood, N., Zhang, X., Rosli, B.H., Gleason, M.L., 2017. First report of Fusarium solani causing fruit rot of loquat (Eriobotrya japonica) in Pakistan. Plant Dis. 101, 839. Booth, C., 1971. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, United Kingdom. Carbone, I., Kohn, L.M., 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 553–556. Chehri, K., Salleh, B., Zakaria, L., 2015. Morphological and phylogenetic analysis of Fusarium solani species complex in Malaysia. Microb. Ecol. 69, 457–471. Coleman, J.J., 2016. The Fusarium solani species complex: ubiquitous pathogens of agricultural importance. Mol. Plant Pathol. 17, 146–158. da Silva-Rocha, W.P., Zuza-Alves, D.L., de Azevedo Melo, A.S., Chaves, G.M., 2015. Fungal peritonitis due to Fusarium solani species complex sequential isolates identified with DNA sequencing in a kidney transplant recipient in Brazil. Mycopathologia 180, 397–401. Farr, D.F., Rossman, A.Y., 2018. Fungal databases, U.S. National fungus collections, ARS, USDA. Retrieved. https://nt.ars-grin.gov/fungaldatabases/. Glass, N.L., Donaldson, G.C., 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61, 1323–1330. Han, M., Lee, H.R., Choi, M.N., Lee, H.S., Lee, S.W., Park, E.J., 2017. First report of Fusarium solani causing soft rot on the tuber of Gastrodia elata in Korea. Plant Dis. 101, 1323-1323. Huang, S.P., Li, Z.L., Wei, J.G., Mo, J.Y., Li, Q.L., Guo, T.X., Yang, X.B., 2017. First report of stem canker caused by Fusarium solani on Tectona grandis in China. Plant Dis. 101, 2148-2148. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 33, 1870–1874. Leslie, J.F., Summerell, B.A., Bullock, S., 2006. The Fusarium laboratory manual (Vol. 2, No. 10). Blackwell Pub, Ames, IA. Lombard, L., Van der Merwe, N.A., Groenewald, J.Z., Crous, P.W., 2015. Generic concepts in Nectriaceae. Stud. Mycol. 80, 189–245. Nalim, F.A., Samuels, G.J., Wijesundera, R.L., Geiser, D.M., 2011. New species from the Fusarium solani species complex derived from perithecia and soil in the Old World tropics. Mycologia 103, 1302–1330. Pandey, A., Juwantha, R., Chandra, S., Kumar, A., Kannojia, P., Khanna, D., Pandey, S., 2018. First report of Fusarium solani causing wilt of Melia dubia. For. Pathol. 48 e12398. Schroers, H.J., Samuels, G.J., Zhang, N., Short, D.P., Juba, J., Geiser, D.M., 2016. Epitypification of Fusisporium (Fusarium) solani and its assignment to a common phylogenetic species in the Fusarium solani species complex. Mycologia 108, 806–819.
Acknowledgments This research was supported by the Youth Foundation of Hunan Educational Committee (grant no. 17B128), Natural Science Foundation of Hunan Province, China (Grant No. 2018JJ3229) and the Hunan Provincial Innovation Foundation for Postgraduates (grant no. CX2018B395).
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Su, H.R., He, H., Huang, Q.Z., Lu, N.H., Zhang, Y.B., 2014. First report of black spot of Acanthus ilicifolius caused by Fusarium solani in China. Plant Dis. 98, 1438-1438. Verde, I., Abbott, A.G., Scalabrin, S., Jung, S., Shu, S., Marroni, F., Zuccolo, A., 2013. The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nat. Genet. 45, 487. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, pp. 315–322. Xie, S.H., Zhang, C., Chen, M.S., Wei, Z.P., Jiang, Y.B., Lin, X., Zheng, Y.Q., 2018. Fusarium proliferatum: a New pathogen causing fruit rot of peach in Ningde, China. Plant Dis. 102, 1858-1858.
Yang, C., Wu, Z.Q., Qi, H., Chen, C.Q., Wang, X., Yang, L.N., Gao, J., 2018. Fusarium solani, a New pathogen causing postharvest fruit rot on kiwifruit (Actinidia deliciosa) in China. Plant Dis. 102, 443-443. Zhang, N., O’Donnell, K., Sutton, D.A., Nalim, F.A., Summerbell, R.C., Padhye, A.A., Geiser, D.M., 2006. Members of the Fusarium solani species complex that cause infections in both humans and plants are common in the environment. J. Clin. Microbiol. 44, 2186–2190. Zheng, L., Peng, Y., Zhang, J., Ma, W.J., Li, S.J., Zhu, T.H., 2015. First report of Fusarium solani causing root rot of Juglans sigllata Dode in China. Plant Dis. 99, 159-159.
174
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Short communication
Fusarium solani causing fruit rot of peach (Prunus persica) in Hunan, China Jun Zi Zhu 1, Chang Xin Li 1, Chao Jun Zhang, Ying Wang, Xiao Gang Li **, Jie Zhong * Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests, Hunan Agricultural University, Nongda Road 1, Furong District, Changsha City, Hunan Province, 410128, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Fusarium solani Prunus persica Fruit rot disease Identification
During 2017 to 2018, a fruit rot of peach (Prunus persica) occurred in Hunan Province of China. The symptoms showed as soft, brown water-soaked lesions with offwhite sporulation, in some cases, on the fruit surface, extending in the flesh, which was decayed. The fungus was isolated and identified as Fusarium solani based on morphological characteristics and molecular identification using the internal transcribed spacer (ITS), a portion of the translation elongation factor-1 alpha (EF-1α) and beta-tubulin gene sequences. The pathogenicity of the isolated F. solani was confirmed. To our knowledge, this is the first report of F. solani causing fruit rot of peach in Hunan, China.
1. Introduction
2. Material and methods
Peach [Prunus persica (L.) Batsch], belonging to the Prunus genus in family Rosaceae, is the third most important deciduous fruit tree in the world. China is the country with the largest peach cultivation area and yield (Verde et al., 2013). Many fungal diseases are commonly wide spread during production, storage and marketing of this fruit. Disease control is vital for the production of peach fruits. Fusarium solani is a species complex (FSSC) frequently isolated from soils, plants, animals, insects and humans that act as decomposers, endophyte, nutritional symbiont and pathogens (Booth, 1971; Schroers et al., 2016). This species represents a phylogenetically and biologically complex group accommodating several formae speciales and biological species. Recently, many molecular identification strategies, such as sequencing of internal transcribed spacer (ITS) regions and translation elongation factor-1α (TEF-1α) have been developed for identification of this species (Nalim et al., 2011; Chehri et al., 2015). During August in 2017 and 2018, fruit rot symptoms were observed in a peach orchard (Cuimitao) in the Hunan Province of China (25.73� N, 112.73� E), with about 50% of fruits infected. The symptoms showed as soft, brown water-soaked lesions with offwhite sporulation, in some cases, on the fruit surface, extending into the flesh, which was decayed. This study was conducted to determine the causal agent of this fruit rot disease of peach via morphological observation, molecular identifica tion and pathogenicity tests.
2.1. Sampling and pathogen isolation During 2017 to 2018, samples of infected fruits displaying soft, brown water-soaked lesions were arbitrarily collected from a peach orchard, and 15 fruit samples from 8 different peach trees were used for fungal isolation. Symptomatic tissues were excised at the border of le sions, surface disinfected with 70% ethanol for 30s, 0.1% HgCl2 for 1.5min, rinsed in sterile distilled water, and then plated onto potato dextrose agar (PDA, amended with 100 mg L 1 streptomycin sulphate) and incubated at 26 � C in the dark. After 2–3 days of incubation, mycelia plugs grown from the infected tissues were transferred to fresh PDA for subculture. Pure cultures were obtained by single conidial isolation. 2.2. Morphological and molecular characteristics A total of 15 isolates were recovered. Since all recovered isolates were identical in colony morphology on PDA, one isolate, TZ4, was used as a representative for further evaluation in morphological and cultural characterization. Mycelium plugs (5 mm in diameter) aseptically picked from the edge of actively growing cultures were transferred on PDA and carnation leaf agar (CLA). The morphology and color of the cultures were examined, and the colony growth rate was assessed in millimeter per day by measuring colony diameters daily for at least 7 days. When
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X.G. Li), [email protected] (J. Zhong). 1 Jun Zi Zhu and Chang Xin Li contributed equally to this work. https://doi.org/10.1016/j.cropro.2019.05.009 Received 11 December 2018; Received in revised form 2 May 2019; Accepted 10 May 2019 Available online 11 May 2019 0261-2194/© 2019 Published by Elsevier Ltd.
J.Z. Zhu et al.
Crop Protection 122 (2019) 171–174
master mix (containing dNTPs), 1 μl (10 μM) of each primer set, 1 μl of genomic DNA (10 ng), and 12 μl of sterile double distilled water. PCR conditions included an initial denaturation at 94 � C for 5 min, followed by 35 cycles of denaturation at 94 � C for 30 s, annealing at 55–57 � C for 1 min, extension at 72 � C for 3 min, and a final extension at 72 � C for 10 min. The PCR products were verified by 1% agarose gel electropho resis and sequenced using Sanger sequencing. Generated sequences were analyzed by Blastn against to publicly available sequences in National Center for Biotechnology Information. Nucleotide sequences were aligned using CLUSTALW implemented in MEGA7 (Kumar et al., 2016) and phylogenetic tree was constructed using neighbour-joining method, with a bootstrap test of 1000 replications. 2.3. Pathogenicity tests Pathogenicity was determined on surface-sterilized matured peach fruits. Mycelial plugs (5 mm in diameter) were taken from a colony actively growing on PDA for five days. The fruits were surfacedisinfected with 75% ethanol for 5 s and then inoculated with myce lial plugs. Sterile agar plugs without mycelial were used as control. All the inoculations were maintained at humid chambers at 26 � C. The tests were repeated twice, with each one having five peach fruits. Symptoms were observed four days after inoculation. Fungi were re-isolated and reidentified from infected fruits showing symptoms to verify Koch’s postulates.
Fig. 1. Symptoms of fruit rot on peach fruits (a) Rot symptom was observed on peach fruits in an orchard; (b) pathogenicity test on wounded peach fruit with mycelial plugs (inoculated) showing symptoms 4 and 10 days post-inoculation; check (CK) remained symptomless.
3. Results and discussion Fruit rot symptoms were observed in a peach orchard in the Hunan Province of China. Soft, water-soaked lesions occurred on the outer surface of the fruit, and then extended to the whole fruit with inner tissues becoming brown and decayed (Fig. 1a). Decayed symptoms were also observed in the pedicle often leading to fruit dropping. From 15 symptomatic fruits collected in the field, fungal strains with similar colony characteristics were consistently isolated and cultured on PDA. Colonies showed a brown pigment in the media center, and sparse or white aerial hypha, with a growth rate of 0.46 cm/day on PDA. In addition, we cultured the fungus on PSA and SNA (Spezieller N€ ahrstoffarmer agar) for morphological identification. As expected, the fungus showed distinct culture morphology when cultured on different mediums, showing lesser pigmentation on PAS medium and thinner hyphae on SNA medium (Fig. 2a). When cultured on Carnation leaf agar (CLA) for 10 days at 26 � C, macroconidia were abundantly produced, cylindrical in shape, 30.4 to 41.3 � 4.4–5.4 μm in size, and with 3–5 transverse septa (n ¼ 50); microconidia were also observed hyaline, oval or fusiform, 6.7 to 15.6 � 3.1–4.7 μm and 0 to 3 transverse septa (n ¼ 50) (Fig. 2b). These morphological and cultural characteristics were consistent with de scriptions of F. solani (Booth, 1971). The pathogen was further confirmed by molecular identification. The gene regions of ITS, EF-1α and beta-tubulin were PCR amplified and sequenced. These sequences were deposited in GenBank (GenBank Accession Nos. MG203887, MG205523 and MG205524 for ITS, EF-1α and beta-tubulin, respectively). BLASTn homology search showed that the ITS, EF-1α and beta-tubulin showed 99% identities to the corre sponding sequences of F. solani strains whose GenBank Accession Nos were available (KF030977.1, DQ247569.1 and KJ720638.1, respec tively) (da Silva-Rocha., 2015; Zhang et al., 2006; Su et al., 2014). Phylogenetic analysis were performed based on multiple alignments of the sequenced ITS, EF-1α and beta-tubulin with other corresponding sequences of Fusarium spp. In each of the three phylogenetic trees, the representative strain TZ4 obtained in this work was clustered with F. solani strains (Fig. 3). Based on the morphological characteristics and molecular identification, this fungus was tentatively identified as F. solani (Mart.) L. Lombard & Crous (Leslie et al., 2006; Lombard et al., 2015).
Fig. 2. Cultural and morphology of Fusarium solani isolate in this study. (a) Colony morphology of the representative isolate, TZ4, cultured on PDA, PSA, and NSA, respectively; (b) Microscopic observation of the macroconidia and microconidia of the F. solani isolate.
cultured on CLA for 10 days at 26 � C, conidia dispersed in water were visualized by a light microscope at 400�magnification. The length and width of at least 50 conidia were measured. For molecular identification, genomic DNA was extracted from mycelium by CTAB method as described previously (White et al., 1990). Internal transcribed spacer (ITS) region of rDNA, a portion of the translation elongation factor-1 alpha (EF-1α) gene and beta-tubulin gene were used as target genes to identify the causal fungal organism (White et al., 1990; Carbone et al., 1999; Glass et al., 1995). Target genes were amplified using specific primer pair sets using PCR, where each reaction was a 50 μl reaction containing 25 μl of a PCR 172
J.Z. Zhu et al.
Crop Protection 122 (2019) 171–174
Fig. 3. Phylogenetic analysis of the F. solani isolate TZ4. Phylogenetic trees were inferred from ITS (a), EF-1α (b) and beta-tubulin gene (c) sequences of F. solani and other Fusarium spp. using the neighbour-joining method by MEGA 7.0. Bootstrap values supporting the branch were showed at the nodes. The scale bar indicated the number of nucleotide substitutions per site.
References
Pathogenicity was determined by placing the mycelial plugs on the surface of matured peach fruits. Four days post inoculation, inoculated fruits showed the decayed symptoms similar to naturally infected orig inal fruits, but the control fruits remained symptomless. After 10 days, nearly one third of surface area of the inoculated fruits rotted on which grey white hypha occurred (Fig. 1b). F. solani was specifically re-isolated and confirmed via morphological characteristics and molecular identi fication from inner tissue of the inoculated fruits, confirming Koch’s postulates. China is the leading producer of peaches worldwide. F. solani is a plant pathogen detrimental to many plants including both agricultural crops and trees (Coleman et al., 2016; Farr and Rossman, 2018). For example, it has been reported to cause symptoms of wilt on Melia dubia (Pandey et al., 2018), fruit rot on Eriobotrya japonica (Abbas et al., 2017), root rot on Juglans sigllata Dode (Zheng et al., 2015), soft rot on the tuber of Gastrodia elata (Han et al., 2017), stem canker on Tectona grandis (Huang et al., 2017) and postharvest fruit rot on kiwifruit (Yang et al., 2018). However, until now, F. solani has not been reported as a pathogen of peach. Since this pathogen could be a potential threat on peach production leading to heavy loss in peach in the Hunan province, appropriate chemical and cultural management strategies should be adopted to control spread. Peach fruit rots caused by other Fusarium species have been identified in China, such as F. proliferatun on peach in Ningde, China (Xie et al., 2018). However, to our knowledge this is the first report of F. solani causing fruit rot of peach in Hunan, China.
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Acknowledgments This research was supported by the Youth Foundation of Hunan Educational Committee (grant no. 17B128), Natural Science Foundation of Hunan Province, China (Grant No. 2018JJ3229) and the Hunan Provincial Innovation Foundation for Postgraduates (grant no. CX2018B395).
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