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Keywords:

  • Capsicum annuum;
  • corynespora blight;
  • multigene phylogenetic analysis;
  • pathogenic variation;
  • target spot

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In order to develop a method for discrimination of Corynespora cassiicola isolates pathogenic to sweet pepper among Japanese isolates, this study analysed pathogenic variations of 64 Japanese isolates of C. cassiicola on perilla, cucumber, tomato, aubergine and sweet pepper, and their multigene phylogeny. Japanese isolates were divided into seven pathogenicity groups (PG1–PG7). The virulence of isolates in PG1–PG5 was restricted to perilla, cucumber, tomato, aubergine and sweet pepper, respectively. Isolates in PG6 were virulent to sweet pepper, tomato and aubergine. Isolates in PG7 were avirulent to all tested plants. Multigene phylogenetic analysis of the isolates based on β-tubulin, translation elongation factor 1-α, calmodulin and actin genes showed three divergent clusters, MP-A, MP-B and MP-C. These clusters included all isolates in PG1, PG2, PG8 and PG9 (MP-A), PG3 and PG5 (MP-B) and PG4 and PG6 (MP-C). Isolates in PG7 were distributed amongst all clusters. Furthermore, random amplified polymorphic DNA (RAPD) analysis using universal primers, Q17 (5′-GAAGCCCTTG-3′) and Q13 (5′-GGAGTGGACA-3′), facilitated discrimination of isolates virulent on sweet pepper amongst isolates in MP-B and MP-C, respectively. Together, a combination of the multigene analysis and the RAPD technique allowed the discrimination of the isolates virulent to sweet pepper.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The plant pathogenic fungus, Corynespora cassiicola, causes target spot on leaves, stems, roots and flowers of more than 280 plant species, including many economically important crops in over 70 countries (Silva et al., 1995). Reports of a new disease caused by the pathogen have increased worldwide, such as corynespora leaf spot on balsam pear (Momordica charantia) in Korea (Kwon et al., 2005), leaf spot on basil (Ocimum basilicum) in Italy (Garibaldi et al., 2007), target spot on highbush blueberry (Vaccinium corymbosum) in Argentina (Hong et al., 2007) and cocoa (Theobroma cacao), papaya (Carica papaya), sweet potato (Ipomoea batatas) and cassava (Manihot esculenta) in Sri Lanka (Silva et al., 2000).

C. cassiicola was once considered a weak pathogen affecting rubber plants (Hevea brasiliensis). However, since the 1980s, the severity of corynespora leaf fall has been increasing in many countries where rubber is a vital commodity. Since rubber clones became more susceptible with time and the susceptibility of rubber clones to the pathogen differed in different geographic regions, it was postulated that C. cassiicola might vary in its pathogenicity (Nghia et al., 2008). Therefore, molecular variations among C. cassiicola isolates, especially isolates affecting rubber plants, were analysed using internal simple sequence repeat (ISSR) molecular fingerprinting analysis (Nghia et al., 2008; Qi et al., 2009), restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) (Silva et al., 1998) and random amplified polymorphic DNA (RAPD) analysis (Silva et al., 1998, 2003) to facilitate the development of rubber clones with enhanced resistance to all genetic clusters of C. cassiicola.

Asexually reproducing fungi do not undergo regular recombination and genetic variation results mainly from the accumulation of spontaneous mutations. In these fungi the whole genome is linked, transmitted as a unit from one generation to the next, so different regions in the genome should share the same evolutionary history (Taylor et al., 1999). The evolution of phenotypic traits in asexual plant-pathogenic fungi, such as host specificity or relatedness among pathogens, can be studied by analysing genealogies of genes that do not have a direct functional relationship to the phenotypes of interest (Jiménez-Gasco et al., 2002).

Analysis of DNA sequences of several mostly single-copy nuclear genes have recently been used to study evolutionary relationships among closely related fungi. The multiple gene genealogies are an effective tool for reconstructing the evolutionary history of pathogenic fungi. β-tubulin, translation elongation factor 1-α (EF-1α), calmodulin and actin genes have been used successfully in delineating relationships between fungi, including phylogenetic studies of plant-pathogenic fungi such as Cylindrocarpon destructans (Seifert et al., 2003) and Fusarium solani f.sp. eumartii (Romberg & Davis, 2007). Phylogenetic analysis based on multiple genes has been reported for many fungal species, such as Verticillium dahliae (Collado-Romero et al., 2008), Pyricularia spp. (Hirata et al., 2007) and Fusarium avenaceum (Nalim et al., 2009). Recently, Dixon et al. (2009) carried out phylogenetic analyses using nucleotide sequences of four genes – the ITS regions of rDNA, two random hypervariable loci and the actin-encoding gene – on 143 isolates of Corynespora spp., and showed a lack of recombination within the species and six statistically significant phylogenetic lineages among the isolates of C. cassiicola that correlated with host of origin, pathogenicity and growth rate, but not with geographic location of collection.

In Japan, C. cassiicola has caused severe damage on perilla (Perilla frutescens), cucumber (Cucumis sativus), tomato (Solanum lycopersicum) and aubergine (Solanum melongena) crops. It was previously reported that corynespora blight occurred on sweet pepper (Capsicum annuum) in Kochi prefecture, Japan (Shimomoto et al., 2008), and has spread through the western areas of Japan, leading to massive losses in sweet pepper production. The exact characterization and understanding of the genetic diversity of isolates of plant-pathogenic fungi are key elements for ecological and epidemiological analysis of the pathogens and developing disease-control systems against them. Previously, pathogenic variation was shown among isolates from Kochi prefecture on perilla, cucumber, tomato, aubergine and sweet pepper (data not shown). However, information regarding phylogenetic and genetic diversity of Japanese isolates of C. cassiicola remains unclear.

In this study, to develop a method for discrimination of isolates virulent to sweet pepper among Japanese isolates of C. cassiicola, based on their pathogenic and genetic variation, we first constructed the pathogenic profiles of Japanese C. cassiicola isolates were first constructed by dividing the isolates into seven groups based on their pathogenicity on perilla, cucumber, tomato, aubergine and sweet pepper. They were then divided into three phylogenetic groups, multigene phylogenetic group (MP)-A, MP-B and MP-C, by multigene phylogenetic analysis based on nucleotide sequences of the β-tubulin, EF-1α, calmodulin and actin genes. Because isolates virulent to sweet pepper were placed in MP-B and MP-C, a specific and sensitive method was developed to discriminate them from other isolates within MP-B and MP-C by RAPD analysis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal isolates

Sixty-four isolates of C. cassiicola from different hosts and geographical locations in Japan, and a Netherlands isolate (CBS162·60), a USA isolate (ATCC64204) and an Indian isolate (ATCC26316) were analysed in this study (Table 1). All isolates were purified with single spores to ensure genetic uniformity and were cultured on potato dextrose agar (PDA; DIFCO Laboratories).

Table 1.   Pathogenicity profiles for Japanese isolates of Corynespora cassiicola
IsolateOriginal hostLocationYear of collectionMPaPGbPathogenicityc
Sweet pepperAubergineTomatoCucumberPerilla
  1. aPhylogenetic groups based on the nucleotide sequences of combinations of multiple genes, β-tubulin, translation elongation factor 1-α, calmodulin and actin.

  2. bPathogenicity groups based on virulence towards sweet pepper, aubergine, tomato, cucumber and perilla.

  3. cWhen expanding lesions in inoculated leaves were observed, the isolate was rated virulent (+). When no lesion or nonexpanding pinpoint lesions in inoculated leaves were observed, the isolate was rated avirulent (−).

ONPerillaKochi, Japan2006A1+
S1PerillaKochi, Japan2004A1+
S2PerillaKochi, Japan2004A1+
SO2PerillaKochi, Japan2006A1+
PC95010PerillaOoita, Japan1995A1+
PC9810-2PerillaAichi, Japan1998A1+
MAFF305093PerillaChiba, Japan1959A1+
CO1CucumberKochi, Japan2006A2+
CU1CucumberKochi, Japan2006A2+
CU2CucumberKochi, Japan2006A2+
K1CucumberKochi, Japan2006A2+
CC3-2CucumberKochi, Japan2005A2+
1-1jppaCucumberKochi, Japan2004A2+
KE1CucumberEhime, Japan2006A2+
KE3CucumberEhime, Japan2006A2+
02-C-ST-H1-2CucumberOkayama, Japan2002A2+
C-KM2-11-2CucumberOkayama, Japan2000A2+
MAFF306176CucumberNagano, Japan1988A2+
1–5CucumberTokushima, Japan2006A2+
4–3CucumberTokushima, Japan2006A2+
MC1CucumberMiyazaki, Japan2006A2+
KSCucumberShimane, JapanUnknownA2+
KI1CucumberIwate, Japan2007A2+
KI5CucumberIwate, Japan2007A2+
CC041125CucumberAichi, Japan2004A2+
MAFF744073CucumberFukuoka, Japan2001A2+
IbCor1481CucumberIbaraki, JapanUnknownA2+
MAFF240444Balsam pearOkinawa, Japan2007A2+
MBBalsam pearMiyazaki, Japan2007A2+
MAFF240443PapayaOkinawa, Japan2007A7
MAFF305087SoyabeanSaitama, Japan1945A7
MAFF305088SoyabeanMie, Japan1950A7
MAFF240792HydrangeaKanagawa, Japan2008A7
Nelumbo04East India lotusTokushima, Japan2004A7
CBS162·60CucumberNetherlands1957A8++++
ATCC64204CucumberUSAUnknownA9+++++
TTRC1-1TomatoKochi, Japan2005B3+
02-T-NS1-3TomatoOkayama, Japan2002B3+
02-T-TD18-3TomatoOkayama, Japan2002B3+
KTOTomatoKagoshima, Japan2007B3+
GCC1TomatoGifu, Japan2001B3+
GCC2TomatoGifu, Japan2001B3+
MT1TomatoMiyazaki, Japan2005B3+
LC93009TomatoOita, Japan1993B3+
LC93020TomatoOita, Japan1993B3+
NBRC100170TomatoTochigi, JapanUnknownB3+
MAFF240205Scarlet sageTokyo, Japan2006B5+
MAFF240206Scarlet sageKanagawa, Japan2006B5+
ATCC26316TomatoIndiaUnknownB6+++
ACC001MandevillaAichi, Japan2007B7
NRC2-1AubergineKochi, Japan2004C4+
N2AubergineKochi, Japan2006C4+
NK-C13AubergineKochi, Japan2006C4+
Shimane aubergine 1AubergineShimane, JapanUnknownC4+
Kurashiki No. 17AubergineOkayama, Japan1999C6+++
PC1-2Sweet pepperKochi, Japan2005C6+++
T3Sweet pepperKochi, Japan2006C6+++
SN1Sweet pepperKochi, Japan2006C6+++
FN3Sweet pepperKochi, Japan2006C6+++
CN1Sweet pepperKochi, Japan2006C6+++
T1Sweet pepperKochi, Japan2006C6+++
T2Sweet pepperKochi, Japan2006C6+++
TKSweet pepperKagoshima, Japan2007C6+++
MAFF240207Sweet pepperMiyazaki, Japan2005C6+++
EN1AubergineKochi, Japan2005C6+++
N1AubergineKochi, Japan2005C6+++
MAFF240496PlumeriaOkinawa, Japan2007C7

Pathogenicity tests

Sweet pepper (cv. Kyonami), aubergine (cv. Senryo No. 2), tomato (cv. Momotaro), cucumber (cv. ZQ-7) and perilla (cv. Aochirimen-Shiso) seedlings were grown in pots containing commercial soil (Tsuchitaro, Sumitomo Forestry) in a greenhouse at 25 ± 5°C for 6 weeks. Inocula were prepared by scraping the mycelium from 10-day-old PDA cultures and suspending them in sterile distilled water (Shimomoto et al., 2008). The resulting spore suspensions were filtered through double layers of cheesecloth, and then adjusted to 104 spores mL−1. The test plants were inoculated with 5 mL spore suspension per plant by spraying. Control plants were treated with sterile distilled water. Eight plants for each of the isolates were used for the pathogenicity tests. Following inoculation, plants were incubated for 48 h under 100% relative humidity at 25°C and then grown in a glasshouse at 25 ± 5°C. Disease symptoms in inoculated leaves were observed for 7 days after inoculation. Isolates were considered virulent when expanding lesions in inoculated leaves were observed, and avirulent when no lesion or nonexpanding pinpoint lesions in inoculated leaves were observed. Nonexpanding pinpoint lesions were certified as the hypersensitive response, because observation under a microscope showed no extension of penetration hyphae in inoculated leaves, which were cut into sections, decolorized with 99% methyl-alcohol, and then stained with 0·1% cotton-blue.

DNA extraction

Mycelium of C. cassiicola covering about 66% of the area of a 9-cm PDA plate was scraped from the surface of the agar with a sterile glass slide. Genomic DNA was extracted as described by Luo et al. (2005).

PCR amplification and sequencing

Primers sets to amplify and sequence the β-tubulin, EF-1α, calmodulin and actin genes are detailed in Table 2. The reaction mixture (20 μL) consisted of primers at 0·4 μm each, dNTPs at 0·2 mm each, 0·5 U Ex Taq (Takara) and 200 ng extracted fungal DNA. Amplification of β-tubulin and calmodulin genes was performed at 94°C for 2 min; followed by 40 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; and a final extension step at 72°C for 7 min. For EF-1α and actin PCRs, the thermal-cycling protocol involved initial denaturation at 94°C for 2 min; 40 cycles at 94°C for 1 min, 58°C for 1 min and 72°C for 1 min; with a final extension at 72°C for 7 min. The PCR-amplified products were purified using a MonoFas DNA refining kit (GL Sciences) according to the manufacturer’s instructions. Sequencing was performed with the same primers used in PCR along with a BigDye Terminator v3·1 Cycle Sequencing kit (Applied Biosystems). Cycle sequencing reaction products were analysed on an ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems) according to the manufacturer’s instruction.

Table 2.   Primers used in this study
GenePrimerSequenceAnnealing temperature (°C)Length of amplicon (bp)References
β-tubulintub-F1tub-R25′-CCTCCAAACCGGTCAATG-3′ 5′-CTGGGTCAACTCGGGGAC-3′55979This study
EF-1αEF1-728FEF1-986R5′-CATCGAGAAGTTCGAGAAGG-3′ 5′-TACTTGAAGGAACCCTTACC-3′58334Carbone & Kohn, (1999)
CalmodulinCAL-228FCAL-737R5′-GAGTTCAAGGAGGCCTTCTCCC-3′ 5′-CATCTTTCTGGCCATCATGG-3′55580–585
ActinACT-512FACT-783R5′-ATGTGCAAGGCCGGTTTCGC-3′ 5′-TACGAGTCCTTCTGGCCCAT-3′58336–348

Phylogenetic analysis

The DNA sequences were edited with genetyx-windows Version 6 (Genetyx Corporation). The nucleotide sequences of the β-tubulin (954 bp), EF-1α (273 bp), calmodulin (465–470 bp) and actin (292–303 bp) genes were aligned and phylogenetic trees were constructed using clustalw (DNA database of Japan; http://clustalw.ddbj.nig.ac.jp/top-j.html) via the neighbour-joining (NJ) method (Saitou & Nei, 1987) using genetic distances computed with Kimura’s two-parameter model (Kimura, 1980). The NJ phylogenetic trees were drawn by treeview (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). The nucleotide sequences of β-tubulin, EF-1α, calmodulin and actin genes of Corynespora smithii isolate NBRC8162, obtained from the NITE Biological Resource Center, Japan, were used as the outgroup for phylogenetic tree reconstructions.

RAPD analyses

Isolates were divided into three multigene phylogenetic groups: MP-A, MP-B and MP-C, by multigene phylogenetic analysis based on nucleotide sequences of the β-tubulin, EF-1α, calmodulin and actin genes. To discriminate isolates virulent to sweet pepper from other isolates in MP-B and MP-C, RAPD analyses were performed using primers P1–P20, Q1–Q20 and W1–W20 (Operon Technologies), for all 14 and 17 isolates in MP-B and MP-C, respectively. PCR amplification was performed using Ex Taq in PCR Thermal Cycler MP programmed at 95°C for 2 min; then 45 cycles at 94°C for 1 min, 35°C for 2 min and 72°C for 3 min; with a final extension at 72°C for 7 min. PCR products were separated by electrophoresis on 1·5% (w/v) agarose gels, stained with ethidium bromide and visualized under UV light. Each amplification was performed at least twice in separate experiments to ensure reproducibility.

Nucleotide sequence accession numbers

Nucleotide sequences of the β-tubulin, EF-1α, calmodulin and actin genes of the 67 isolates used in this study were assigned DDBJ accession numbers AB539168AB539439.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Pathogenicity test

The virulence of 64 Japanese isolates of C. cassiicola was tested alongside three foreign isolates, on perilla, cucumber, tomato, aubergine and sweet pepper. Seven isolates from perilla showed virulence on perilla only (Table 1). Twenty isolates from cucumber and two from balsam pear were virulent on cucumber. Ten isolates originally from tomato demonstrated virulence on tomato only. The virulence of four isolates from aubergine was restricted to aubergine, but a further two isolates that were virulent on aubergine were also harmful to tomato and sweet pepper. Nine sweet-pepper and one tomato isolate from India also showed virulence on aubergine, tomato and sweet pepper. Two isolates from scarlet sage (Salvia splendens) showed virulence only on sweet pepper. Seven isolates from papaya, soyabean (Glycine max), hydrangea (Hydrangea macrophylla), East India lotus (Nelumbo nucifera), mandevilla (Mandevilla sp.) and plumeria (Plumeria rubra) were avirulent to all tested plants, although they were reportedly virulent on isolated plants (http://www.gene.affrc.go.jp/databases-micro_pl_diseases_en.php). These groups were thus designated pathogenicity group (PG) 1 to PG7. The virulence of isolates in PG1–PG5 was restricted to perilla, cucumber, tomato, aubergine and sweet pepper only, respectively. Isolates in PG6 were virulent to tomato, aubergine and sweet pepper. Isolates in PG7 were avirulent to any tested plants. An isolate from the Netherlands (CBS162·60), isolated from cucumber, was virulent on cucumber, tomato, aubergine and sweet pepper (PG8). A USA isolate (ATCC64204) was virulent on all tested plants (PG9) (Table 1).

Multigene analysis using nucleotide sequences of β-tubulin, EF-1α, calmodulin and actin genes

Dixon et al. (2009) reported a low level of sequence variation of the ITS regions of rDNA within C. cassiicola isolates from American Samoa, Brazil, Malaysia and the USA. To detect genetic variation, the housekeeping genes, β-tubulin, EF-1α, calmodulin and actin, were analysed in Japanese C. cassiicola isolates. The NJ phylogenetic tree generated by combining the four protein-coding genes placed the 67 isolates, including the 64 Japanese isolates, in three major clusters, designated MP-A, MP-B and MP-C, with strong bootstrap support (Fig. 1). MP-A included all isolates of PG1 and PG2, MP-B included all isolates of PG3 and PG5, and MP-C contained all isolates of PG4 and PG6. The PG7 isolates were divided into three clusters. Both CBS162·60 (PG8) and ATCC64204 (PG9) were grouped into the MP-A cluster, and ATCC26316 (PG3) was included in MP-B.

image

Figure 1.  Phylogenetic tree of Corynespora cassiicola Japanese isolates based on the combined nucleotide sequences of β-tubulin, translation elongation factor 1-α, calmodulin and actin genes, constructed with clustalw (the DNA Data Bank of Japan; http://www.ddbj.nig.ac.jp/search/clustalw-j.htlm) using the neighbour-joining (NJ) method. Scale bar indicates genetic distance, i.e. the expected number of substitutions per position. The number shown next to each node indicates the percentage bootstrap values of 1000 replicates that exceeded 80%. Figures in parentheses indicate pathogenicity groups (PGs) based on virulence towards sweet pepper, aubergine, tomato, cucumber and perilla: (1) PG1, (2) PG2, etc. Nucleotide sequences of β-tubulin, translation elongation factor 1-α, calmodulin and actin genes from Corynespora smithii NBRC8162 were used as the outgroup for tree construction.

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RAPD analysis of MP-B and MP-C isolates

The pathogenicity test showed that isolates in PG5 were virulent only on sweet pepper. On the other hand, isolates in PG6 were virulent on sweet pepper, but also on aubergine and tomato. Phylogenetic analysis showed that isolates in PG5 and PG6 were included in MP-B and MP-C, respectively. These results showed that sequencing of the four protein-coding genes did not allow discrimination of isolates virulent to sweet pepper among Japanese isolates. To discriminate isolates in PG5 and PG6 from the MP-B and MP-C clusters, RAPD analysis was performed using 60 universal primers. RAPD analysis using Q17 (5′-GAAGCCCTTG-3′) could discriminate isolates virulent on sweet pepper among isolates in MP-B. Furthermore, RAPD analysis using Q13 (5′-GGAGTGGACA-3′) showed that isolates of MP-C could be divided into three subgroups correlated with their pathogenic diversity (Fig. 2). However, RAPD analysis using Q17 and Q13 was not able to facilitate discrimination of MP-C and MP-B isolates, respectively in relation to their pathogenicity groups (data not shown).

image

Figure 2.  Random amplified polymorphic DNA (RAPD) patterns using universal primers, Q17 (5′-GAAGCCCCTTG-3′) and Q13 (5′-GGAGTGGACA-3′), generated from 14 isolates (a) and 17 isolates (b) of Corynespora cassiicola included in multigene phylogenetic group (MP)-B and MP-C, respectively. (a) Lanes 1–14, isolates of C. cassiicola: 1, TTRC1-1; 2, 02-T-NS1-3; 3, 02-T-TD18-3; 4, KTO; 5, GCC1; 6, GCC2; 7, MT1; 8, LC93009; 9, LC93020; 10, NBRC100170; 11, MAFF240205; 12, MAFF240206; 13, ATCC26316; 14, ACC001. (b) Lanes 15–31, isolates of C. cassiicola: 15, NRC2-1; 16, N2; 17, NK-C13; 18, Shimane aubergine 1; 19, Kurashiki No. 17; 20, PC1-2; 21, T3; 22, SN1; 23, FN3; 24, CN1; 25, T1; 26, T2; 27, TK; 28, MAFF240207; 29, EN1; 30, N1; 31, MAFF240496. Lane M, DNA Ladder marker. PG3, PG4, PG5, PG6 and PG7 are pathogenicity groups of the isolates, based on virulence towards sweet pepper, aubergine, tomato, cucumber and perilla.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Pathogenicity tests play an important role in the identification of the species and races of a fungus. It has been reported that some isolates of C. cassiicola show virulence on a wide range of hosts, whereas others exhibit host specificity (Spencer & Walters, 1969; Cutrim & Silva, 2003; Pereira et al., 2003). As many as eight different pathogenicity profiles were recognized among 28 isolates collected from soyabean in Mexico, cucumber in Florida and a diverse range of hosts in Nigeria (Onesirosan et al., 1974). Dixon et al. (2009) demonstrated at least 16 unique pathogenicity profiles within 50 isolates of C. cassiicola on the eight crop plants basil, bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), cucumber, papaya, soybean, sweet potato and tomato. Furthermore, Dixon et al. (2009) and Onesirosan et al. (1974) demonstrated that isolates virulent to tomato were likely to be virulent on several other hosts. In the present study, isolates from aubergine were divided between PG4 and PG6. Furthermore, two isolates from balsam pear were virulent on cucumber (PG2) and two isolates from scarlet sage were virulent on sweet pepper (PG5). This would suggest that grouping isolates based on the original host is problematic for predicting pathogenicity and genetic relatedness. However, isolates from perilla, cucumber and tomato were virulent only to their respective hosts. The isolates in PG6 from sweet pepper and aubergine were an exception, demonstrating pathogenicity on sweet pepper, aubergine and tomato. Therefore, the pathogenic variability among Japanese isolates might be smaller than that of isolates from other countries.

Molecular genetic techniques for the determination of mutations in living organisms have been shown to be important tools for the analysis of the genetic diversity and epidemiology of fungal plant pathogens (Maclean et al., 1993). Dixon et al. (2009) demonstrated that the lack of correlation between phylogenetic data and geographic location of C. cassiicola isolates collected from 68 different plant species in American Samoa, Brazil, Malaysia, Micronesia and the USA provides sufficient evidence to indicate a recent global movement of isolates from all six phylogenetic lineages, and that geographically diverse isolates from the same host plant often share identical haplotypes, potentially indicating a degree of host specialization. They also demonstrated that haplotypes determined by the nucleotide sequences of the ITS regions of rDNA are congruent with the phylogenetic lineages (PL) in the four-locus combined analysis (Dixon et al., 2009). In the present study, results of phylogenetic analysis on isolates from perilla, cucumber, tomato, aubergine and sweet pepper suggests a correlation between the original host and phylogenetic lineages among Japanese isolates. Furthermore, each isolate in MP-A and MP-B showed pathogenicity on only one plant species among perilla, cucumber, tomato and sweet pepper. Therefore, the phylogenetic diversity amongst the Japanese isolates was also responsible for their pathogenicity on perilla, cucumber, tomato, aubergine and sweet pepper. All this data also supported the lack of pathogenic diversity in the Japanese isolate of C. cassiicola, especially from these plants. Therefore, it is postulated that a progenitor strain of C. cassiicola may have been systematically spread throughout the Japanese agricultural system in the past, possibly by the dissemination and importation of live plant materials such as seeds and seedlings.

Multigene analysis based on the nucleotide sequences of these housekeeping genes did not allow discrimination of isolates virulent to sweet pepper among Japanese isolates, showing that the degree of resolution was not sufficiently discriminatory to determine the diversity in pathogenicity among the Japanese isolates virulent to sweet pepper. It was thus thought that the evolutionary rate of pathogenicity of sweet-pepper-pathogenic isolates was greater than the mutation rate of the housekeeping genes. RAPD analysis has also been employed and revealed significant polymorphism among groups of C. cassiicola isolates, exhibiting a correlation between RAPD groups and the features of the isolates, such as pathogenicity, geographical origin and host-plant genotype from which the isolate was collected (Silva et al., 1995, 1998, 2003). The present research suggests that a combination of multigene analysis based upon nucleotide sequences of the housekeeping genes and RAPD analysis allows for the specific discrimination of Japanese C. cassiicola isolates that are pathogenic to sweet pepper. This technique could lead to a better understanding of their ecology and ways in which disease-control systems can be developed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank T. Miyamoto, N. Miyake, K. Tanina, K. Hirota, T. Kuroki, Y. Kushima, T. Kamikado, N. Mashita, N. Kirino, and Drs S. Nekoduka, H. Otani, M. Ueno H. Watanabe, and M. Nao for their gifts of fungal isolates. We also thank Drs H. Otani and M. Kusaba for their valuable suggestions and Dr Y. Tosa for his critical review. This research was supported by the NIAS Genebank Project of the National Institute of Agrobiological Sciences.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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