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20Russian Journal of Nematology, 2011, 19 (2), 173 - 180
Characterisation of Meloidogyne species on Southern Herbs in Hainan island using perineal pattern and esterase phenotype and amplified mitochondrial DNA restriction fragment length polymorphism analysis
Mei-ying Fu, Mian-cai Chen, Tong-bin Xiao and Hui-fang Wang
'Institute of Environment & Plant Protection, Hainan Academy of Agricultural Sciences, Hainan Key Laboratory for Control of Plant Diseases and Insect Pests , Haikou, 571100, China;
e-mail: [email protected].
Accepted for publication 12 October 2011
Summary. Various kinds of southern herbs have been planted in Hainan island because of the suitable climatic conditions. However, root-knot nematodes have become a serious problem to cultivation of southern herbs. Thirty Meloidogyne populations on southern herbs from Hainan island were characterised in terms of perineal pattern and esterase phenotype, and Hinfl and EcoRl was used to double digest the amplified products from the mitochondrial DNA (mtDNA) region between the COII and lrRNA genes. Perineal patterning and esterase phenotyping revealed that 17 of the populations were M. incognita, five were M. javanica, three were M. arenaria, four were M. enterolobii and one was M. hispanica. ln order for rapid and reliable identification of Meloidogyne species, we used the primers C2F3 and 1108 to amplify the intergenic region between COII and lrRNA genes of mtDNA, and Hinfl and EcoRl were used to double digest the amplified products. The results showed that five Meloidogyne species can be distinguished. It is concluded that mtDNA-PCR-RFLP was a rapid and reliable approach for molecular identification of common Meloidogyne species.
Key words: Hainan, nematode identification, mtDNA-PCR-RFLP, root-knot nematodes, southern herbs.
Southern herbs are cultivated in Southeast Asia and Africa as medicinal material. Various kinds of southern herbs have been planted in Hainan island because of the suitable climatic conditions. According to several surveys, there were more than 30 kinds of southern herbs planted in Hainan island. For example, pepper, betel nut, morinda citrifolia, cinnamon, sandalwood, clove, Amomum villosum and others have been planted there on a large-scale (Yan, 2001). According to the statistics, the harvested area of betel nut were 20,733.3 ha, with the total output of 6.43 million metric tons and output value of 1.54 billion yuan in 2005. However, the damage caused by root-knot nematodes (Meloidogyne spp.) has a serious impact on yield and quality of southern herbs.
Root-knot nematodes are economically important plant pathogens, and more than 90 nominal species have been described. These root endoparasites are widely distributed from the tropical south to the
temperate north, infecting many crop species and causing crop losses up to 70% in China (Xu et al, 1994). Meloidogyne incognita, M. javanica, M. arenaria, M. chitwoodi, M. fallax and M. hapla accounted for more than 95% of the occurrences of this genus and are the most widespread species. In China, M. incognita, M. javanica, M. arenaria and M. hapla cause serious economic losses, and the former three are distributed mainly in tropical regions, while the latter occur in cool temperate regions.
Effective management of root-knot nematodes often requires rapid and accurate species identification of target nematode populations. Meloidogyne species are traditionally differentiated from each other by morphological characters and isozyme phenotypes. Morphological identification demands considerable skill and could be unreliable due to significant intraspecific morphological variations in Meloidogyne spp. Isozyme
phenotyping has been shown to be a valuable means for precise identification of major Meloidogyne species (Esbenshade & Triantaphyllou, 1985a, b; Carneiro et al, 2000). In particular, esterase and malate dehydrogenase are frequently used. However, isozyme analysis is only performed with single females, not single second-stage juveniles (J2), males or eggs (Esbenshade & Triantaphyllou, 1990). Since the female stage is often unavailable in soil samples, the isozyme method requires the time and space to establish and maintain populations in culture from single egg masses in order to obtain females. Based on the polymerase chain reaction (PCR), developing methods to identify Meloidogyne species using individual single juvenile has been the objective of various studies. Several molecular methods which detect DNA polymorphisms between species have been used with DNA extracted from J2; for example, five major species of Meloidogyne were discriminated by amplification and restriction of the intergenic region between the COII and lrRNA genes in the mitochondrial genome (Powers & Harris, 1993). Hinfl was used to digest the amplification products from the mtDNA region between the COII and lrRNA genes, which enabled identification of five Meloidogyne species from China (Xu et al, 2004). Specific SCAR primers were designed to identify M. incognita, M. javanica and M. arenaria, which enabled identification of these species from single J2, and three PCR reactions were obtained from extracts of single J2 of these species (Meng et al., 2004).
The aim of this study was to research perineal patterns and esterase (Est) phenotypes and mtDNA-PCR-RFLP patterns of 30 Meloidogyne populations collected from Hainan of China. Our objectives were to determine the species identity of the populations and assess the utility of mtDNA polymorphism for reliable diagnosis of major root-knot nematodes occurring in Hainan.
MATERIAL AND METHODS
Nematode populations. Thirty Meloidogyne populations used in this study were listed in Table 1. All the populations were derived from single eggmasses from field populations and three single eggmasses were hand-picked from one field population. The original field populations were randomly collected from infected southern herbs in Hainan Island of China. Nematodes were routinely maintained on tomato at 20-28°C in a glasshouse.
Perineal pattern slice. Perineal pattern temporary slides were made, using the method described by Taylor and Netscher (1974) with minor modifications. The infected roots were placed in
0.9% sodium chloride solution and the females were dissected out from the root under the dissecting microscope. The females were transferred to 45% lactic acid in a plastic Petri dish and the posterior end cut off with a very small scalpel. The perineal patterns were trimmed and transferred to water on a microscope slide, and a cover slip was applied. Perineal patterns are formed by expansion and alteration of the juvenile body and retain the lateral lines, the tail tip, and the phasmids.
Esterase analysis. Est were analysed according to the procedure of Esbenshade and Triantaphyllou (1985a) with minor modifications. The separating and stacking gels were homogeneous 7% and 4% polyacrylamide, respectively. Protein extracts from 20 females were loaded into the same lane. The bridge buffer was Tris/glycine pH 8.3. After electrophoresis, the gels were stained for Est for 20 min. Designation of Est phenotypes followed the scheme of Esbenshade and Triantaphyllou (1985b).
DNA preparation. Nematode egg-masses were hand-picked from infected tomato roots and J2 were hatched in water at 25°C Total genomic DNA was then extracted from ca 10 ^l of packed J2 using the method of Cenis (1993). The quality and quantity of extracted DNA samples were checked by subjecting them to electrophoresis and Gold View staining together with uncut ADNA standards. For PCR amplification from single J2, individual J2 were hand-picked and ruptured with a steel needle in a 6 ^l drop of lysis buffer (1*ExTaq PCR buffer 100 ^g ml-1 proteinase K; TaKaRa Biotech, Dalian, China) on a glass slide. Nematode lysates were then transferred to PCR tubes and frozen for 30 min at -80°C. After freezing, samples were incubated for 1 h at 60°C and 15 min at 95°C before being processed for PCR amplification.
PCR-RFLP procedure. Primers C2F3 (5-GGTCAATGTTCAGAAATTTGTGG-3 ) and 1108 (5'-TACCTTTGACCAATCACGCT-3') (Powers & Harris, 1993) were used to amplify the intergenic region between the COII and lrRNA genes in the mitochondrial genome of Meloidogyne. PCR was conducted in a 25 ^l reaction volume containing10 ng of purified DNA or 5 ^l of crude J2 lysate, 0.2 ^M of each primer, 1* Ex Taq PCR buffer, 2 mM Mgcl2, 0.2 mM of each dNTP and 1 U Ex Taq DNA Polymerase (Sangon, Shanghai, China). For amplification using purified DNA, reaction mixtures were subjected to a preheating at 94°C for 4 min, followed by 35 cycles of 1 min at 94°C, 1 min at 52°C and 2 min at 70°C, and a final incubation at 72°C for 5 min using a Mastercycler Personal thermal cycles. For amplification using crude J2 lysates, the number of cycles was raised to 40.
Tablel. Meloidogyne populations used in this study
Species Populations Origin Primary host Est mtDNA-PCR-RFLP
PCR product (bp) HinfI and EcoRl digestion (bp)
M. incognita MIWN1 Wanning, Hainan Morinda citrifolia I2 1700 1200, 400, 100
MIWN2 Wanning, Hainan Morinda citrifolia I2 1700 1200,400,100
MIDZ1 Danzhou, Hainan Morinda citrifolia I2 1700 1200, 400, 100
MIHK1 Haikou, Hainan Morinda citrifolia I2 1700 1200,400,100
MIHK2 Haikou, Hainan Morinda citrifolia I2 1700 1200, 400, 100
MIDA Dingan, Hainan Morinda citrifolia I2 1700 1200,400,100
MIWN3 Wanning, Hainan Morinda citrifolia I2 1700 1200, 400, 100
MITC Tunchang, Hainan Fructus alpiniae oxyphyllae I2 1700 1200, 400, 100
MIWN4 Wanning, Hainan Fructus alpiniae oxyphyllae I2 1700 1200, 400, 100
MILS Lingshui, Hainan Fructus alpiniae oxyphyllae I2 1700 1200, 400, 100
MIWN5 Wanning, Hainan Fructus alpiniae oxyphyllae I2 1700 1200, 400, 100
MIHK3 Haikou, Hainan Catharanthus roseus I2 1700 1200, 400, 100
MIWC1 Wenchang, Hainan Catharanthus roseus I2 1700 1200,400,100
MIWC2 Wenchang, Hainan Catharanthus roseus I2 1700 1200, 400, 100
MIWN6 Wanning, Hainan Alpinia officinarum I2 1700 1200, 400, 100
MIHK4 Haikou, Hainan Betel nut I2 1700 1200, 400, 100
MIDZ2 Danzhou, Hainan Clove I2 1700 1200, 400, 100
M. javanica MJWC Wenchang, Hainan Catharanthus roseus J3 1700 1200, 500, 400, 100
MJTC Tunchang, Hainan Aquilaria J3 1700 1200, 500, 400, 100
MJHK Haikou, Hainan Catharanthus roseus J3 1700 1200, 500, 400, 100
MJDA1 Dingan, Hainan Morinda citrifolia J3 1700 1200, 500, 400, 100
MJDA2 Dingan, Hainan Morinda citrifolia J3 1700 1200, 500, 400, 100
M. arenaria MAWN1 Wanning, Hainan Morinda citrifolia A2 1100 1100
MADZ Danzhou, Hainan Morinda citrifolia A2 1100 1100
MAWN2 Wanning, Hainan Morinda citrifolia A2 1100 1100
M. hispanica MHDZ Danzhou, Hainan Morinda citrifolia S2-M1 1700 1200, 500
M. enterolobii MEWC Wenchang, Hainan Aquilaria VS1-S1 700 700
MEHK1 Haikou, Hainan Aquilaria VS1-S1 700 700
MEHK2 Haikou, Hainan Morinda citrifolia VS1-S1 700 700
MEWN Wanning, Hainan Clove VS1-S1 700 700
Fig. 1. Perineal patterns of five Meloidogyne species. A: MIWN1 (M. incognita); B: MJWC (M. javanica); C: MADZ (M. arenaria); D: MHDZ (M. hispanica); E: MEWC (M. enterolobii).
Photograph of perineal pattern A (M. incognita): Elongated with more or less flattened dorsal arch. Striae smooth to wavy and with no lateral lines. Perineal pattern B (M. javanica): Perineal pattern with the definite incisures on the lateral lines separating the striae of the dorsal and ventral sectors. They lead to the tail terminus. Dorsal arch rounded to flattened. Perineal pattern C (M. arenaria): Striae wavy obvious with same forking at lateral lines and short irregular striae near lateral lines. Perineal pattern D (M. hispanica): Perineal pattern with folds along both lateral lines. Striae rounded to flattened. Striae of the dorsal arch into a square. Perineal pattern E (M. enterolobii): Perineal pattern with no lateral lines. Striae smooth to wavy and have high dorsal arch.
Following amplification, 5 pl of PCR products were separated on a 1.0% agarose gel, stained with Gold View, and visualised under UV illumination. Using HinfI and EcoRI to double digestion of amplified products was conducted in a 20 pl volume containing 5 pl of PCR product, 2 pl of 10*restriction buffer H and 1 pl of each enzyme (Hinfl and EcoRI) (TaKaRa Biotech, Dalian, China) and 11 pl sterile water. Double digestion was allowed to proceed for 6 h at 37°C. Restricted products were assayed on a 1.5% agarose gel.
RESULTS
Perineal pattern. Large variations were found in root-knot nematode perineal patterns. Even perineal pattern of different populations of the same root-knot nematode was very different. In this study, we have listed typical perineal patterns of five Meloidogyne species (Fig. 1).
Fig. 2. Est phenotypes of some populations of Meloidogyne incognita, M. javanica, M. arenaria, M. enterolobii and M. hispanica from Hainan island. 1: MJWC (Est phenotype J3); 2: MJTC (J3); 3: MJHK (J3); 4: MIWN1 (I2); 5: MIWN2 (I2); 6: MEWC (VS1-S1); 7: MEHK1 (VS1-S1); 8: MHDZ (S2-M1); 9: MEHK2 (VS1-S1); 10: MEWN (VS1-S1); 11: MJDA1 (J3); 12: MIDZ1 (I2); 13: MIHK1 (I2); 14: MIDA (I2); 15: MAWN1 (A2).
Fig. 3. PCR products amplified from representative populations of Meloidogyne incognita, M. javanica, M. arenaria, M. enterolobii and M. hispanica from Hainan Province using primers C2F3 and 1108. 1: MJWC; 2 MJTC; 3: MJHK; 4: MJDA1; 5: MIWN1; 6: MIWN2; 7 MIDZ1; 8: MEWC; 9: MEHK1; 10: MHDZ; 11 MAWN1; 12: MADZ; 13: MIDA; M: DL2000 DNA marker (TaKaRa Biotech, Dalian China).
MtDNA-PCR-RFLP analysis. Using mtDNA primers C2F3 and 1108, an amplification product was obtained from either purified DNA or crude J2 lysate from each of the populations listed in Table 1. Fragments of three different sizes were amplified (Table 1, Fig. 2). Specifically, the populations of M. incognita, M. javanica and M. hispanica had a PCR product of 1700bp; the M. arenaria populations produced a smaller product of 1100bp and the M. enterolobii populations produced a product of 700bp (Fig. 3). When the amplification products were double-digested at the same time with the restriction enzyme HinfI and EcoRI, distinctive enzyme phenotype-correlated RFLP profiles were generated
that allowed the differentiation of the five species studied (Fig. 4). Different restriction sites on the 1700bp product separated M. javanica, M. incognita and M. hispanica from each other. Four fragments of 1200, 500, 400 and 100bp were generated in all the M. javanica populations; three fragments of 1200, 400 and 100bp were produced in all the M. incognita populations; and two fragments of 1200 and 500bp occurred in the M. hispanica. The 700bp fragment characteristic of M. enterolobii and 1100bp fragment characteristic of M. arenaria were not restricted by HinfI and EcoRI.
Fig. 4. HinfI and Ecoffl-restriction patterns of PCR products amplified from representative populations of Meloidogyne incognita, M. javanica, M. arenaria, M. enterolobii and M. hispanica from Hainan island using primers C2F3 and 1108. 1: MJWC; 2: MJTC; 3: MIWN1; 4: MIWN2; 5: MEWC; 6: MEHK1; 7: MHDZ; 8: MAWN1; 9: MADZ; M: DL2000 DNA marker (TaKaRa Biotech, Dalian China).
DISCUSSION
The species identity of 30 Meloidogyne populations collected on southern herbs from Hainan island in China was revealed by perineal patterning and esterase phenotyping and confirmed by mtDNA-PCR-RFLP analysis in the present study. Meloidogyne incognita, M. javanica, M. arenaria, M. enterolobii and M. hispanica were identified by 56.7, 16.7, 10,13.3 and 3.3% of the total samples, respectively. Meloidogyne incognita was the predominant species on southern herbs from Hainan island in China. Meloidogyne enterolobii was rare in other regions of China, although it has been reported on distributing in Hainan island and Guangdong province (Xu, 2004; Zhuo, 2008). Meloidogyne hispanica was first reported on papaya from Hainan island in 2007 (Wang, 2007), and in this study it was found on Morinda citrifolia from Hainan island in China.
Since isozyme phenotypes have been shown to be reliable criteria for identification of major Meloidogyne species (Esbenshade & Triantaphyllou, 1985b), we used the traditional Est phenotype and perineal pattern as the means of identification in this study. Five root-knot nematodes were identified from the populations on seven southern herbs with the different Est phenotypes (i.e., I2, J3, A2, S2-M1, VS1-S1) and perineal patterns. One populaition has been identified as M. hispanica, its Est phenotype was S2-M1; moreover, it was only found from Hainan island in China. Meloidogyne enterolobii was originally found infecting pacara earpod trees in Hainan island of China (Yang & Eisenback, 1983). It has so far been recorded only from Hainan island (Zhao, 2000; Xu, 2004) and Guangdong Province (Zhuo, 2008). Meloidogyne enterolobii was regarded as a considerable threat to agriculture because it is able to reproduce on many crops, e.g., tomato, potato, Cajanus cajan, Psidium guajava, Capsicum annuum, Vigna sinensis and others. In this paper, four populations have been identified as M. enterolobii on Morinda citrifolia, aquilaria and clove, its Est phenotype is VS1-S1. However, previous studies have shown that M. mayaguensis had female perinean pattern, host range and cytogenetic features similar to those of M. enterolobii (Yang & Eisenback, 1983; Esbenshade & Triantaphyllou, 1985; Rammah & Hirschmann, 1988). In addition, populations of M. mayaguensis showed an Est phenotype (VS1-S1) identical to that of M. enterolobii (Esbenshade & Triantaphyllou, 1985; Fargette & Braaksma, 1990; Xu et al, 2004). Meloidogyne mayaguensis was differentiated from
M. enterolobii only by some minor morphological differences and by a different malate dehydrogenase phenotype (N3c in M. mayaguensis vs N1a in M. enterolobii). Interestingly, the sequence comparison revealed that the 662bp (data not shown) sequence between primers C2F3 and 1108 for M. enterolobii in the present study was identical to that reported for M. mayaguensis. The mtDNA sequence evidence presented here suggests that M. mayaguensis could be conspecific with M. enterolobii, as it was soon suggested by Hunt & Handoo (2009). The taxonomic relationship of the two species needs to be studied in more detaily in the future.
With the increase in multiple cropping, damage caused by root-knot nematodes is getting worse. Effective control and management often requires rapid and accurate species identification of target nematode populations. Based on the extensive application of PCR technology, the mtDNA-PCR-RFLP assay has been used to diagnose the major Meloidogyne species in recent studies. Powers and Harris (1993) designed primers C2F3 and 1108 to amplify the intergenic region between the mitochondrial COII and lrRNA genes to discriminate five major Meloidogyne species. HinfI restriction patterns of the C2F3 and 1108 amplification products from M. incognita and M. javanica have been reported in several studies with different results (Powers & Harris, 1993; Williamson et al., 1994; Orui, 1998; Blok et al., 2002; Sun et al., 2005). While Powers and Harris (1993) found three restriction fragments of 1000, 400, 300bp for M. incognita, and two fragments of 1000 and 700bp for M. javanica, the other investigators reported two products of 1300 and 400bp for M. incognita, and no restriction of the 1700bp product for M. javanica. We have not found anyone using two restriction enzymes to digest root-knot nematodes in previous studies. In this study, we used the HinfI and EcoRI to double digest at the same time for 30 Meloidogyne species, and the results showed that all populations of M. incognita can be digested into three restriction fragments of about 1200, 400 and 100bp, almost all the populations of M. javanica can be digested into four restriction fragments of about 1200, 500, 400 and 100 bp, the population of M. hispanica can be digested into two restriction fragments of about 1200 and 500 bp, and the PCR product of specific region on mtDNA of M. arenaria and M. enterolobii cannot be digested. All the populations of M. javanica have two restriction sites on 1700 bp fragments, and it has four restriction fragments because two restriction enzymes were used to digest randomly. Different restriction sites on the 1700bp
product separated M. javanica, M. incognita and M. hispanica from each other. It concluded that mtDNA-PCR-RFLP is a rapid and reliable approach for molecular identification of common Meloidogyne species.
ACKNOWLEDGEMENT
This work was funded by the Special Fund for Agro-scientific Research in the Public Interest of China (201103018).
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«южных трав» острова Хайнань по данным морфологии перивульварной пластинки и RFLP mtDNA. Резюме. Различные виды целебных т.н. «южных трав» культивируются на о-ве Хайнань. В последнее время значительной проблемой их выращивания стали галлообразующие нематоды. Тридцать популяций Meloidogyne с «южных трав» Хайнаня были исследованы методом изучения перивульварной платинки, спектров эстераз, а также при помощи рестрикции ферментами Hinfl и EcoRl ПЦР-продукта - участка митохондриальной ДНК (mtDNA) между генами COII и lrRNA. Изучение перивульварных пластинок и фенотипы эстераз показали, что 17 популяций представляют собой M. incognita, пять - M. javanica, три - M. arenaria, четыре - M. enterolobii и одна - M. hispanica. Для быстрого определения видов Meloidogyne использовали праймеры C2F3 и 1108 для амплификации участка mtDNA между генами COII и lrRNA, который затем подвергали двойной рестрикции в помощью Hinfl и EcoRl. Результаты этого анализа выявляют присутствие 5 видов Meloidogyne. Метод mtDNA-PCR-RFLP - быстрый и надежный подход к определению видов Meloidogyne.
