Genetic diversity of Fusarium mangiferae isolated from mango malformation disease in China

Scientia Horticulturae 165 (2014) 352–356

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Genetic diversity of Fusarium mangiferae isolated from mango malformation disease in China Feng Liu a,b , Ji-guang Wei a,∗ , Ru-lin Zhan b , Xiong-chang Ou b , Jin-mei Chang b a

College of Agriculture, Guangxi University, Nanning, Guangxi, China South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Key Laboratory of Tropical Fruit Biology, Ministry of Agriculture, Zhanjiang, Guangdong, China b

a r t i c l e

i n f o

Article history: Received 13 September 2013 Received in revised form 20 October 2013 Accepted 14 November 2013 Keywords: Mango malformation Fusarium mangiferae Genetic diversity Vegetative compatibility group Inter simple sequence repeat

a b s t r a c t Mango malformation disease, caused by Fusarium mangiferae, is one of the most important diseases affecting mango production. Thirty-eight isolates from MM-affected mango tissues were collected from two cities and assessed for genetic diversity using vegetative compatibility groups (VCGs) and inter simple sequence repeats (ISSRs). VCGs were determined among all isolates, using nitrate non-using (nit) mutants. Out of the 455 nit mutants generated, 78.02% used both nitrite and hypoxanthine (nit1), 13.63% used hypoxanthine but not nitrite (nit3) and 7.91% used nitrite but not hypoxanthine (NitM). Five different VCGs were identified by pairing the mutants on nitrate medium. Seventy-two bands were amplified by 14 ISSR primers, with an average of 5.14 bands per primer. The size of the amplified products ranged from 300 to 2000 bp. At a genetic similarity of 0.76, the unweighted pair-group method with arithmetic mean analysis separated the isolates into five distinct clusters, comprising 33, 2, 1, 1 and 1 isolates. The VCG and ISSR data support the conclusion that the isolates of F. mangiferae are not necessarily related by geographic origin, type of host tissue, or the mango cultivar from which they were isolated. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Mango malformation is one of the most destructive diseases of Mangifera indica, occurring in most mango-producing regions worldwide. The disease is characterized by malformation of vegetative growth and inflorescences, causing serious yield losses (Ploetz et al., 2002). The vegetative form of the disease is observed most frequently on young seedlings. The auxiliary or apical buds produce misshapen shoots, and have shortened internodes and brittle leaves, that are significantly smaller than those of healthy plants. The major symptoms of inflorescence malformation include abnormally branched and thickened panicles that produce up to three times the normal number of flowers. These flowers are enlarged and do not bear fruit (Zheng and Ploetz, 2002). This disease was first reported in India in 1891 (Kumar et al., 1993), and has subsequently been reported from Egypt, USA, South Africa, Brazil, and China (Youssef et al., 2007; Lima et al., 2009a; Kvas et al., 2008; Zhan et al., 2012). The etiology of the malformation has been a contentious issue. A wide range of biotic and abiotic factors have been reported to cause the disease, including viruses, mites (Sternlicht and Goldenberg, 1976), and nutritional deficiencies (Abo-El Dahab, 1977; Minessy et al., 1971). Convincing evidence that fungi cause

∗ Corresponding author. Tel.: +86 7592859311. E-mail address: [email protected] (J.-g. Wei). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.11.025

the malformation has been in the literature for decades. Fusarium spp. have been the fungal pathogens most commonly associated with the disease since 1966. These include: F. mangiferae, F. proliferatum, F. sacchari, F. sterilihyphosum F. mexicanum, F. tupiense and F. subglutinans, all of which belong to the Gibberella fujikuroi species complex (Leslie, 1993). Some of these species are economically important plant pathogens (Steenkamp et al., 2000; Lima et al., 2009al; Khaskheli et al., 2008; Rodríguez-Alvarado et al., 2008; Otero-Colina et al., 2010). Vegetative compatibility has been widely documented among several fungi. It has proved to be a powerful tool in determining fungal genetic diversity, particularly for species in the genus Fusarium, which were divided into several groups based on their ability to form stable heterokaryons (Puhalla, 1985; Leslie and Summerell, 2006; Smith-White et al., 2001). ISSR technology is sensitive to considerable levels of genetic diversity, classifying fungal populations into different clusters based on the similarity of their coefficients. ISSR analysis is a PCR-based method that involves amplification of a DNA segment at an amplifiable distance between two identical microsatellite repeat regions oriented in opposite directions. It has the advantages of low cost and high efficiency compared with other DNA genotyping techniques (Reddy et al., 2002; Baysal et al., 2010; Bogale et al., 2005). Mango malformation disease was first observed in China in 1992, with a disease incidence rate of 75.4% in Yuanjiang county of Yunnan province (Chen, 1992). So far, the disease only occurred in the city of Panzhihua in Sichuan province and

F. Liu et al. / Scientia Horticulturae 165 (2014) 352–356 Table 1 Characterization of Fusarium mangiferae isolates based on VCG and ISSR patterns. Isolate numbera

Mango cultivar

Tissue

VCGb

ISSR pattern

MG01 MG02 MG03 MG04 MG05 MG06 MG07 MG08 MG09 MG10 MG11 MG12 MG13 MG14 MG15 MG16 MG17 MG18 MG19 MG20 MG21 MG22 MG23 MG24 MG25 MG26 MG27 MG28 MG29 MG30 MG31 MG32 MG33 MG34 MG35 MG36 MG37 MG38

Keitt Keitt Keitt Keitt Hongxiangya Hongxiangya Keitt Zill Hongxiangya Keitt Hongxiangya Keitt Keitt Zill Hongxiangya Keitt Zill Keitt Hongxiangya Keitt Keitt Zill Keitt Zill Keitt Keitt Keitt Sannian Macheso Tainong No. 2 Tainong No. 2 Sensation Keitt Keitt Keitt Sensation Hongxiangya Sannian

Floral Floral Floral Floral Floral Vegetative Floral Floral Vegetative Floral floral Vegetative Floral Floral Floral Floral Floral Floral Floral Floral Floral Floral Floral Floral Floral Floral Floral Floral Vegetative Floral Vegetative Vegetative Vegetative Vegetative Floral Floral Floral Floral

VCG1 VCG1 VCG3 VCG1 VCG2 VCG1 VCG1 VCG1 VCG1 VCG1 VCG1 VCG1 VCG1 VCG1 VCG1 VCG1 VCG4 VCG1 VCG1 VCG1 VCG4 VCG1 VCG1 VCG1 VCG1 VCG3 VCG1 VCG1 VCG2 VCG1 VCG2 VCG1 VCG5 VCG1 VCG1 VCG1 VCG1 VCG1

B-IV B-IV B-IV B-IV B-IV B-IV B-II B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-IV B-III B-IV B-IV B-IV B-IV B-IV B-IV B-IV A B-IV B-III B-I B-IV B-IV

a Isolates MG01-26 was come from Panzhihua city, Sichun Province, and MG27-38 was from Huapin county, Yunnan Province. b VCG and ISSR are mean of the vegetative compatibility group and inter simple sequence repeats, respectively.

Huaping County in Yunnan province, with incidences of 30–95%. In China, F. mangiferae was confirmed as the main cause of the disease by satisfying Koch’s postulates (Zhan et al., 2012). However, there has been no study of the genetic diversity of the main causal agent of mango malformation in China. The aim of the present study was to (i) assess the genetic diversity of F. mangiferae using VCG and ISSR; (ii) estimate the relationships among isolates with respect to geographic origin, type of tissue affected, and the mango cultivar. 2. Materials and methods 2.1. Fungal isolates Samples were collected from mangoes with either floral or vegetative malformation symptoms from the city of Panzhihua in Sichuan province and Huaping County in Yunnan province. For isolation, samples were cut into 5-mm-long pieces, which were dipped in ethyl alcohol (75%) for 5–10 s, and then surface sterilized with HgCl2 solution (0.1%) for 1 min. the samples were then washed twice with sterile distilled water, dried with blotting paper, and then placed on potato dextrose agar (PDA) medium. Ten tissue pieces were excised from each sample and incubated for 5 days at 28 ◦ C. Isolates were purified by single conidial subcultures on tap water agar. Twenty-six isolates were collected from Panzhihua and the remainder from Huaping (Table 1). Morphological examination of isolates grown on PDA or carnation leaf agar was performed

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according to Leslie and Summerell (2006). All isolates were verified as F. mangiferae using the species-specific primer (Zheng and Ploetz, 2002; data not shown). The isolates identified as F. mangiferae were transferred to SNA medium (per liter, 1 g of KH2 PO4 , 1.0 g KNO3 , 0.5 g of MgSO4 ·7H2 O, 0.2 g of KCl, 0.2 g of dextrose, 0.2 g of sucrose, and 18 g of agar in sterile distilled water) and stored at 4 ◦ C during the period of the experiments. 2.2. Pathogenicity test All isolates of F. mangiferae were screened for their ability to induce symptoms of malformation on mango seedlings. One-yearold mango seedlings planted in pots (10 l capacity) in a glasshouse were used for inoculation. Conidial suspensions were obtained by adding sterile water to the culture plates, mixing the suspension, and then filtering it through two layers of sterile cheesecloth. Wound inoculation was performed by injecting 200 ␮l of conidial suspensions (1 × 106 spores per ml) of each isolate into axillary or apical buds. Five plants were inoculated per isolate. Waterinoculated plants served as controls. The experiment was repeated once. 2.3. Vegetative compatibility test Isolates of F. mangiferae were classified according to their vegetative compatibility group using the method described by Puhalla (1985). In brief, nitrate non-using mutants were generated by placing isolates on a chlorate medium (PSA + 3% KClO3 ). For each isolate, 10 Petri dishes (90 mm × 15 mm) were inoculated with three agar plugs per plate (30 mycelial plugs per isolate) and incubated at 28 ◦ C for 10–15 days. Rapidly growing sectors were transferred to a minimal medium (MM: 30.0 g sucrose, 1 g KH2 PO4 , 0.5 g MgSO4 ·7H2 O, 0.5 g KCl, 0.01 g FeSO4 ·7H2 O, 15 g agar, and 0.2 ml trace element solution in 1000 ml of distilled water [5 g citric acid, 5 g ZnSO4 ·7H2 O, 1 g Fe(NH4 )2 (SO4 )2 ·6H2 O, 0.25 g CuSO4 ·5H2 O, 0.05 g MnSO4 ·H2 O, 0.05 g H3 BO4 , 0.05 g NaMoO4 ·2H2 O, and 95 ml distilled water]) with 3 g nitrate per liter. Sectors that grew well were discarded as putative chlorateresistant, nitrate-using mutants (Klittich and Leslie, 1988), while those that showed thin mycelial growth was retained as putative nit mutants (Leslie and Summerell, 2006). nit mutants were further tested for their ability to use nitrite (MM plus 0.5 g/l NaNO2 ) and hypoxanthine (MM plus 0.2 g/l hypoxanthine) as sole nitrogen sources. Mutants able to use both nitrite and hypoxanthine were classified as nit1, those able to use nitrite but not hypoxanthine were classified as NitM, and those that could use hypoxanthine but not nitrite were classified as nit3 (Klittich and Leslie, 1988; Zheng and Ploetz, 2002). At least two different nit mutants were obtained from each strain and complementary pairings of nit1, nit3, and NitM were made to establish initial self-compatibility (Correll et al., 1989). Mutants in different phenotypic classes were paired on MM with nitrate as the sole nitrogen source. Complementation reactions between NitM and nit1 and/or nit3 mutants of different isolates were repeated twice. Strains that showed prototrophic growth when paired were in the same vegetative compatibility group (VCG), while those lacking prototrophic growth were in different VCGs (Leslie, 1993). VCGs were established by pairing mutants in all possible pairwise combinations. 2.4. DNA extraction For DNA extractions, all isolates of F. mangiferae were grown on half-strength potato dextrose broth in sterile 250 ml conical flasks for 7 days at 25 ◦ C. Mycelia were collected on two layers of cheesecloth, washed in sterile distilled water three times, dried with sterile filter paper, and then ground in to a fine powder in

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Table 2 Details of primers used in this study and the polymorphism of generated bands among Fusarium mangiferae isolates. Primer number

Primer sequencea

Annealing temperature, ◦ C

808 809 812 816 818 835 847 851 855 856 880 881 885 891 Total

(AG)8 C (AG)8 G (GA)8 A (CA)8 T (CA)8 G (AG)8 YC (CA)8 RC (GT)8 YG (AC)8 YT (AC)8 YA GGA GAG GAG AGG AGA GGG GTG GGG TGG GGT BHB (GA)7 HVH(TG)7

55 55 55 50 50 50 60 58 55 55 50 50 58 55

a

Amplified bands Total no of bands generated

Polymorphic bands

6 3 8 5 5 6 7 4 5 6 5 3 6 3 72

5 2 6 3 4 4 7 3 3 4 3 2 4 2 52

B = T/G/C, D = A/T/G, Y = G/C, H = A/T/C, V = A/G/C, R = A/T.

liquid nitrogen. The DNA was extracted using an E.Z.N.A.® HP Fungal DNA Kit (Omega Bio-Tek, Norcross, Georgia), according to the manufacturer’s instructions. The DNA was visualized on a 1% agarose gel (w/v) stained with ethidium bromide and viewed under UV light. DNA concentration and quality were determined with a UV-2450 spectrometer (Shimadzu, Kyoto, Japan) and visualized on agarose gels. The working concentration of DNA was adjusted to 25 ng/␮l and stored at −20 ◦ C. 2.5. PCR amplification of ISSRs Forty ISSR primers with di- or tri-nucleotide repeats were designed based on the nucleotide sequences reported by the University of British Columbia (UBC, http://www.biotech.ubc. ca/services/naps/primers/Primers.pdf). To select primers that produced higher numbers of polymorphic bands for the characterization of F. mangiferae isolates, 40 ISSR primers were screened using DNA samples of five representative isolates from different VCGs based on earlier research. Fourteen primers that generated clear, repeatable and polymorphic bands were selected for PCR amplification of the genomic DNA of all F. mangiferae isolates (Table 2). The PCR volume was 25 ␮l, and included 10 ng template DNA, 1 U TaKaRa Taq DNA polymerase, 0.2 mmol/l primers, 200 mmol/l each of dNTP and 10 × PCR buffer (Mg2+ plus). The reaction program was as follows: 1 cycle at 94 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 30 s, 50–60 ◦ C (depending on primers used) for 30 s, and 72 ◦ C for 2 min. All PCR amplifications were performed at least twice for each isolate. The amplified products were separated on 2% agarose gels and stained with ethidium bromide. Images were photographed and captured by Gel Doc 2000TM (Bio-Rad, Hemel Hempstead, Hertfordshire). 2.6. Statistical analysis The Numerical Taxonomy and Multivariate Analysis System (NT-SYS) software package, Version 2.1 (Rohlf, 2002), was used to analyze the data. Amplified products were scored for the presence (1) or absence (0) of bands and binary matrices were assembled for the ISSR markers. The data matrix was then used to calculate genetic similarity (GS) values between each two strains, according to Nei and Li’s (1979) method: GS = 2Nij /(Ni + Nj ), where Nij is the number of ISSR bands in common between accessions i and j, and Ni and Nj are the total number of ISSR bands observed for accessions i and j, respectively. Cluster analysis was performed using the unweighted pair-group method with arithmetic mean (UPGMA).

3. Results 3.1. Pathogenicity Koch’s postulates were completed successfully with all tested isolates of F. mangiferae in a replicated experiment. Typical vegetative malformation disease symptoms were observed after 12 weeks. No disease symptoms developed among the control plants. 3.2. Vegetative compatibility groups There were 484 chlorate-resistant sectors produced on KClO3 containing potato sucrose agar medium from 38 single-spore isolates. Each isolate of F. mangiferae produces a mean of 11–25 sectors. The isolates differed considerably in their sectoring frequency, with a range of 0.524–1.191 sectors per colony. The majority of the sectors appeared during the first and second week of incubation, with a few appearing in the third week. A total of 455 sectors grew as thin, expansive colonies with no aerial mycelium. This indicated their inability to use nitrate as a sole source of nitrogen. They were designated as nit mutants and comprised 94% of the total chlorateresistant sectors. All nit mutants produced wild-type growth on a complete medium. Among the 455 nit mutants, 355 (78.02%) were able to use nitrite and hypoxanthine (nit1), 62 (13.63%) used hypoxanthine but not nitrite (nit3), and 36 (7.91%) used nitrite but not hypoxanthine (NitM). When nit1 mutants were paired with either nit3 or NitM mutants on nitrate medium, all 38 strains were self-compatible. When mutants from different isolates were paired on a nitrate medium, five different VCGs were identified. Isolate MG33 was incompatible with all others isolates and constituted a unique VCG (VCG5). Groups VCG2, VCG3, and VCG4 each contained three (MG05, MG29, and MG31), two (MG03 and MG26) and two (MG17 and MG21) isolates, respectively. The remaining 30 strains were compatible with the other isolates and were grouped in VCG1 (Table 1). 3.3. ISSR analysis The ISSR amplification of total genomic DNA from the 38 isolates produced two to seven bands. Among these were a number of polymorphic bands, ranging from one to six. The size of the amplified products ranged from of 300 to 2000 bp. Seventy-two amplification products were produced with an average of 5.14 bands per primer (Table 2). Among these, 52 (72%) were polymorphic. An example of the amplification reactions with primer 847 is presented in Fig. 1. A similarity matrix based on the proportion of shared fragments

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Fig. 1. Representative ethidium bromide-stained agarose gels showing the amplification products generated from Fusarium mangiferae isolates with primers 847. Lanes 1–38 show products resulting from PCR reactions with DNA extracted from isolates MG01 to MG38, respectively, as templates. Lane M contains the molecular weight markers.

was used to establish the level of relatedness between the 38 F. mangiferae isolates. The genetic similarities between isolates ranged from 0.5972 to 0.9862. Isolates MG16 and MG17 were the most similar (0.9862 genetic similarity), whereas isolates MG07 and MG33 were the most distinct (0.5972) and were grouped in separate clusters. Isolate MG33 proved to be the most distinct genetically. The dendrogram consisted of two major clusters, A and B (Fig. 2), at a genetic distance of 0.69. Cluster A contained only one isolate, MG33, from VCG5, and cluster B contained all the remaining 37 isolates from the other four VCGs. In cluster B, four sub-clusters (sub-clusters I to IV) were observed. Isolates MG35 and MG25 were grouped in sub-cluster III. Sub-cluster IV contained the majority of the isolates (33 out of 38) and VCGs belonging to VCG1, VCG2, VCG3 and VCG4, with the genetic similarities of 0.6944–0.9861. Subcluster III contained isolate MG25 from Sichuan and MG35 from Yunnan. Sub-clusters I and II contained one isolate each (Fig. 2). 4. Discussion Vegetative compatibility is a good indicator of clonal ancestry and is a predictor of genotypic similarity among isolates. In present study, the 38 isolates were divided into five VCGs, indicating high variability within F. mangiferae. However, no clear relationships were revealed among isolates for geographical regions, type of tissues affected, or host cultivar. Some isolates from the same types of tissues affected segregated into different VCGs, such as isolates MG32, MG34, and MG33 from malformed vegetative tissues. MG32

and MG34 were grouped in VCG1, while MG33 segregated into VCG5. Thus, the clusters do not represent isolates form different host tissue. Similarly, there was no correlation between VCGs and cultivar. For example, isolates belonging to VCG1 were from cultivar Keitt, Hongxiangya, Zill, Sannian, Tainong No. 2, and Sensation. Whereas, isolates MG01, MG03, MG21, and MG33 were obtained from cultivar Keitt but were segregated into different VCGs. Similar results were obtained by molecular analysis. Based on the ISSR assay, F. mangiferae isolates from the same geographic region, type of tissue, or cultivar appeared in different clusters. Conversely, isolates in the same clusters were collected from different geographic regions, types of tissues affected, or cultivars. Considering the diverse agricultural environments where from we obtained the isolates, the results suggest that geographic isolation, ecological conditions, and host cultivar do not have a significant effect on the distribution of F. mangiferae genotypes. Although the two techniques both showed high levels of genetic diversity and separated the isolates into five groups, the VCG and ISSR groups did not correspond to each other. Isolates from the same VCGs appeared in different ISSR clusters. Conversely, isolates belonging to the same clusters were also collected from different VCGs. Isolates that fell within the same VCG produced different banding patterns. The F. mangiferae isolates were classified into five groups by both of VCG and ISSR methods. Among them, isolate MG33 is similar to the other isolates, which may be explained by the fact that this strain has the most distant genetic relationship compared with the others. However, based on the VCG and ISSR data analysis, F.

Fig. 2. Dendrogram based on the ISSR similarity matrix data by unweighted pair group method, with average (UPGMA) cluster analysis of isolates for Fusarium mangiferae.

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mangiferae isolates from the same geographic region, type of tissues or mango cultivar from which they were isolated appeared in different groups, which agrees with the results obtained by other molecular methods such as RAPD and AFLP on mango malformation disease pathogens (Iqbal et al., 2006; Zheng and Ploetz, 2002). The high level of genetic diversity observed in this study may be associated with genetic drifts, and mutations within fungal populations, or may be the result of the reproductive strategies of the fungus (Thangavelu et al., 2012; Lima et al., 2009b). The present study was performed to highlight genetic characteristics of F. mangiferae isolates recovered from different mango growing areas in China. PCR-based techniques provided not only a better understanding of variability within F. mangiferae, but also simplified the identification of the pathogen. The results will contribute to the development of disease management methods against mango malformation disease, provide more insight for breeding studies on resistant resources and analysis suggests recombination in this population, compared with clonal reproduction in other populations outside the center of origin of mango. Acknowledgements This research is supported by the Fundamental Research Funds of National Nonprofit Research Institute for South Subtropical Crops Research Institute, CATAS (1630062012008, 1630062013008), the National Natural Science Foundation of China (No. 31270076), and the Plans for Construction of Scientific Topnotch and Innovation Team in Guangxi University. We thank Prof. T.C. Harrington working in Iowa State University for revising the paper. References Abo-El Dahab, M.K., 1977. Correcting malformation symptoms of mango tree in Egypt by soil application of iron chelates. Egypt. J. Phytopathol. 7, 97–99. Baysal, Ö., Siragusa, M., Gümrükcü, E., Zengin, S., Carimi, F., Sajeva, M., Jaime, A., da Silva, T., 2010. Molecular characterization of Fusarium oxysporum f. melongenae by ISSR and RAPD markers on eggplant. Biochem. Genet. 48, 524–537. Bogale, M., Wingfield, B.D., Wingfield, M.J., Steenkamp, E.T., 2005. Simple sequence repeats markers for species in the Fusarium oxysporum complex. Mol. Ecol. Notes 5, 622–624. Chen, X.H., 1992. Mango malformation and control. Pract. Technol. 6, 5–6 (in Chinese). Correll, J.C., Klittich, C.J.R., Leslie, J.F., 1989. Heterokaryon self incompatibility in Gibberella fujikuroi (Fusarium moniliforme). Mycol. Res. 93, 21–27. Iqbal, Z., Mehboob-ur-Rahman Dasti, A.A., Saleem, A., Zafar, Y., 2006. RAPD analysis of Fusarium isolates causing mango malformation disease in Pakistan. World J. Microbiol. Biotechnol. 22, 1161–1167. Khaskheli, M.I., Pathan, M.A., Jiskani, M.M., Wagan, K.H., Soomro, M.H., Poussio, G.B., 2008. First record of Fusarium nivale (Fr.) Ces. association with mango malformation disease (MMD) in Pakistan. Pak. J. Bot. 40, 2641–2644.

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