Int.J.Curr.Microbiol.App.Sci (2019) 8(9): 1987-1998
International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 09 (2019) Journal homepage: http://www.ijcmas.com
Original Research Article https://doi.org/10.20546/ijcmas.2019.809.230
Characterization of Guava Root Knot Nematode, Meloidogyne enterolobii Occurring in Tamil Nadu, India
1 1* 1 2 3 P. Suresh , K. Poornima , P. Kalaiarasan , S. Nakkeeran and R. M Vijayakumar
1Department of Nematology, 2Department of Plant Pathology,3Department of Fruit Crops, Tamil Nadu Agricultural University,Coimbatore, Tamil Nadu-641003, India
*Corresponding author
ABSTRACT
Root knot nematodes (Meloidogyne spp.) are the most common and destructive plant parasitic nematode group with wide host range of crops, including guava (Psidium guajava K e yw or ds L.). Meloidogyne enterolobii (Syn:M. mayaguensis), the root knot nematode of the guava tree, belongs to the group of tropical root knot nematodes and is considered as one of the Guava, root knot most damaging species, due to its wide host range, pathogenicity and ability to develop nematode, and reproduce on several crops carrying resistance genes. The present study was aimed at Meloidogyne enterolobii , identifying Meloidogyne species attacking guava orchards in three districts such as perineal pattern, Dindigul, Coimbatore and Thiruvannamalai districts of Tamil Nadu. The morphological ITS and PCR. study based on perineal patterns of the females confirmed the presence of Meloidogyne enterolobii. In addition, the molecular identification was carried out based on the partial Article Info sequence of internal transcribed spacer (ITS1-ITS2) regions. The amplicon size was
Accepted: approximately 600 bp. The sequences were compared with those of M. enterolobii in the
20 August 2019 GenBank database with high similarity (98%). Sequences were submitted in NCBI and
Available Online: obtained gene accession numbers of Dindigul populations (MK940246 and MK955348),
10 September 2019 Coimbatore populations (MK955350 and MN006626) and Thiruvannamalai populations
(MN381161 and MN381109) were obtained. Phylogenetic studies placed present study population with other Meloidogyne species retrieved from the GenBank database.
Introduction (Perry and Moens, 2006). They are dispersed worldwide and parasitizes nearly every Root knot nematodes, Meloidogyne spp. species of both cultivated and uncultivated constitute one of the major important group of plants. The four major species of Meloidogyne plant parasitic nematodes causing severe viz., M. incognita, M. javanica,M. arenariaand damage to both agricultural and horticultural M. haplaare of immense economic importance crops. They are sedentary endoparasites and because of their wide geographical their parasitic life depends on the success to distribution and wide host range (Perry et al., induce feeding sites in the roots of host plants 2009). The most common species are 1987
Int.J.Curr.Microbiol.App.Sci (2019) 8(9): 1987-1998 estimated to be able to infest more than 5500 major Meloidogyne species including resistant plant species (Trudgill and Blok, 2001) and cotton, sweet potato, tomatoes (Mi-1 gene), estimated yield losses of 25-50% over potato (Mhgene), soybean (Mir1 gene), bell cultivated plants (Taylor and Sasser, 1978). pepper (N gene), sweet pepper (Tabasco gene) and cowpea (Rkgene) is major concern (Yang A large number of crops worldwide are &Eisenback, 1983; Fargette & Braaksma, affecting by root knot nematodes and so far 1990: Brito et al., 2007:Cetintas et al., 2008). more than 100 species have been described (Hunt and Handoo, 2009). Infested plants Precise identification of different species of show poor growth and wilt due to nutrient Meloidogyne is important for the management partitioning alterations and limited water of nematodes (Cenis, 1993). The accurate uptake due to deformations of conducting identification based on a combination of vessels (Kaloshian et al., 1996). Damage is several methods such as morphological more pronounced in tropical climates than in characteristics and morphometrics, host temperate climates because of the favourable preferences biochemical and molecular conditions for nematode survival and techniques are essential methods for multiplication (De Waele and Elsen, 2007 and confirming the species (Eisenback et al., Kaloshianet al., 1996). 1981). Earlier, root knot nematodes were characterized mainly based on morphological Root knot nematode, M. enterolobii (Syn: M. features, such as perineal patterns and mayaguensis) Yang & Eisenback (1983) is morphometric data of second stage juveniles, one of the most important nematode causing male and females (Jepson, 1987; Carneiro and severe problems in guava orchards of India Cofcewicz, 2008). However, morphological especially in Tamil Nadu. Moreover it is and morphometric data require skilled person associated with other soilborne plant and thus may not be sufficient to differentiate pathogens such as Fusarium and cause disease closely related Meloidogyne species complex on guava (Poornima et al., 2016). M. (Hirschmann, 1986; Zijlstra et al., 2000). enterolobii became economically important nematode and they are emerged as major DNA based identification of species is an parasitic nematode in many crops worldwide attractive solution as it does not rely on (Moens et al., 2009). Similar to M. incognita, expressed gene products, and is independent M. enterolobiiis also considered to be highly of environmental influence and life cycle polyphagous species with wide host range stages and has high discriminating power (Yang & Eisenback, 1983). The many hosts (Zijlstraet al., 2000). Different regions DNA include vegetables, tomato, pepper, markers that aid for identification of watermelon (Yang & Eisenback, 1983; Meloidogyne species include the rDNA small Rammah & Hirschmann, 1985), guava subunit (SSU) 18S (Powers, 2004), large (Gomes et al., 2012), ornamental plants (Brito subunit (LSU) 28S D2-D3 expansion et al., 2010) and weeds (Rich et al., 2009). segments (Chen et al., 2003), intergenic spacer (IGS) (Blok et al., 1997), internal M. enterolobii was considered as a highly transcribed spacer (Powers and Harris, 1993) aggressive species and induce severe root galls and mitochondrial DNA (Powers and Harris, than other species of root knot nematodes. 1993 and Xu et al., 2004), random amplified Moreover, heavy infested field became polymorphic DNA (RAPD) (Cenis, 1993) and unviable for guava cultivation in Brazil sequence characterized amplified regions (Carneiro et al., 2007).In addition, the ability (SCAR) markers (Zijlstra et al., 2000). of M. enterolobii in breaking resistance to the 1988
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Therefore, the main goal of the present study Molecular identification of M. enterolobii was to identify the root knot nematode, Meloidogyne enterolobii collected from DNA was extracted from females of root knot different guava orchards of various places in nematodes as per the method described by Tamil Naduthrough morphological, Cenis (1993) with slight modifications. Ten to morphometric and by using molecular tools. twenty females were collected from guava roots (originated from single eggmass) and Materials and Methods placed in 1.5 ml of Eppendorf tube, disinfested with 500 μl of 0.5 % sodium Survey and occurrence of Meloidogyne hypochlorite for 3-5 minutes and centrifuged enterolobii at 10000 rpm for 10 minutes. The pellet was washed with sterile water. Then, 400 μl of Soil and root samples were collected from extraction buffer (250 mMTris-HCl, pH 8.0; guava orchards of different places (Table 1) 250 mM sodium chloride; 50 mM EDTA and such as Ayakudi and old Ayakudi of Dindigul 0.5 % Sodium DodicylSulfate, SDS and 10 μl district, Vettavalam and Aavur of of β-Mercaptoethanol) were added and Thiruvannamalai district and Karamadai and crushed with the help of micro pestle and Thondamuthur of Coimbatore district of Tamil mortarmanually for 10-15 minutes. Then, the Nadu. Collected disease materials (galled homogenate were kept at -20º C for 30 roots) were brought to Department of minutes after the addition of 0.5 volume of Nematology, Tamil Nadu Agricultural 3M sodium acetate (pH 5.2). Tubes were then University Coimbatore for examination. centrifuged at 10,000 rpm for 5 minutes and Infested root were examined in stereo zoom supernatant was transferred to another sterile microscope and then stained with hot acid Eppendorf tube. Two volumes of cold fuchsin and destained with lactophenol for isopropanol were added into tubes and detailed examination. incubated at -20º C for overnight for precipitation of DNA. After incubation, the Morphological identification of M. tubes were centrifuged at 12000 rpm for 10 enterolobii minutes and discarded the supernatant. The pellet was washed with 70% ethanol, air dried Generally, identification of Meloidogyne spp. for one hour at room temperature and was done by studying perineal pattern of resuspended in 50 μl of nuclease free water for females. Perineal patterns of ten egg laying further use. The quality of the DNA was matured females from each location were analysed by 0.8 % of agarose gel prepared as per the procedure described by electrophoresis. Eisenback et al. (1981). Polymerase chain reaction and DNA Second stage juveniles were collected from sequencing soil by Cobb‟s decanting and sieving (Cobb, 1918) method followed by Baermann‟s funnel After DNA extraction, PCR was performed techniques (Schindler, 1961). for 25μlcontains, 1 μl genomic DNA (50 ng), Photomicrograph of perineal patterns, anterior 0.5 μM primers (forward and reverse) , 0.5 and posterior regions of male and females and mM of each dNTPs, 2.5 μl 1× PCR buffer, 2.5 second stage juveniles were made by camera mM MgCl2, and 1.25μl Taq DNA polymerase attached to compound microscope. (Sigma, USA) for amplification of Internal transcribed spacer regions of root knot nematodes. 1989
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The internal transcribed spacer 1 (ITS1) -5.8S high dorsal arch with absence of lateral fields gene was amplified using the forward primer and absence of wings. However, dorsal arch in TW81 (5′ GTTTCCGTAGGTGAACCTGC- perineal patterns were quite variable (Fig 2). 3′) and reverse primer AB28 (5′ - ATATGC Perineal pattern from some females showed a TTAAGTTCAGCGGGT-3‟) Subbotin et al. high trapezoidal dorsal arch similar to that of (2001). M. incognita (Fig 2). In the present study, observed characters coincided with original The PCR cycles began with an initial description given by Yang & Eisen back denaturation at 94°C for 5 min(35 cycles with (1983). Moreover, Thiruvannamalai denaturation for 60 sec at 94°C, primer population slightly differed from the original annealing for 30 sec at 60°C and extension for description (Fig.2), which might be due to 60 sec at 72°C) and final extension at 72°C for change in varietal (of the host) and edaphic 5 min. PCR products were visualized with conditions. Morphology of perineal patterns UV illumination after staining with ethidium have been the typical characteristic used for bromide of 1.2 % agarose gels under a UV the identification of the most common transilluminator (Alpha imager EC 1200) Meloidogyne species since 1949 (Chitwood (Sambrook et al., 1989). Amplified PCR 1949). In the present study, M. enterolobii products were sequenced at Chromous were identified based on perineal patterns and Biotech PVT Ltd, Bangalore, Karnataka. other morphological characters.
DNA sequences were aligned using The morphometrics of M. enterolobii females CLUSTAL W followed by manual recorded an average body length and stylet adjustment. Sequences were deposited in length of all isolates as590 (554-690) μm and NCBI and accession numbersobtained. 14.37 (12.50–16.00) μm (Table 1).The Phylogenetic tree was constructed using average body length and stylet length of males MEGA 7 from aligned sequences of the were 1593 (1175-1742) and 23.33 (22 - present study and retrieved from the 24.5μm). The average body length of the GenBankdatabase. second stage juveniles was 448.33 (415 – 495) and stylet length was 13 (12-13.5μm). Those Results and Discussion observed measurements were similar to the original descriptions given by Rammah and Survey and occurrence of Meloidogyne Hirschmann, 1988. Meloidogyne enterolobii enterolobii was first reported in roots of Enterolobium
All the collected samples were found to be contortisiliquum L. in South China (Yang and highly infested by root knot nematode, M. Eisenback, 1983), while M. mayaguensis was enterolobii. Totally six isolates were isolated first reported on eggplants (Solanum from guava orchards in Dindigul, Coimbatore melongena L.) in Puerto Rico (Rammah and Thiruvannamalai districts. Females and andHirschmann, 1988). second stage juveniles were extracted from root and soil and identified as M. enterolobii The nematode, M. enterolobii was first with the help of morphological characters. reported in 2004 in the Paluma variety of guava, which is the most common variety in Morphological characterization of M. commercial orchards at Limoeiro do enterolobii NorteCounty (Torres et al., 2005) in the eastern region of the state. The female, perineal patterns were round to
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Table.1 Morphometrical measurements of Meloidogyne enterolobii populations
S. Characters Dindigul population* Coimbatore population* Thiruvanamalai population*
No J2 Male Female J2 Male Female J2 Male Female 1 Body length 448.33 1564.00 590 434.00 1585.33 596 457.33 1484.33 589 (415 -495) (1426- (567 -710) (405 - (1448- (577 -740) (435 - (1396- (560 -713) 1650) 476) 1750) 465) 1650) 2 Stylet length 12.00 46.00 544 11. 45 540 11.50 46.33 546 (11 - 14) (40 – 46.00) (395 - 529) (11 - (40 - 47) (405 - 519) (10 - (39 - (395 - 609) 13) 16) 47.00) 3 Body width 23.33 17.00 22.00 17.16 20.33 16.00 (22 -24.5) (14 - 19) (22 -25) (14 - 19) (19 -24) (13 - 19) 4 Tail length 52.8 50.8 48.33 (42 - 62.5) - - (45-62) - - (40-66) - - 5 Vulval slit 31.66 29.00 30.33 length (PCP) - - (26 -33) - - (22-39) - - (26 -33) *Values are mean of five replications
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Fig.4 Maximum likelihood tree after an alignment of consensus sequences based on the ITS region of various Meloidogyne enterolobii identified in this study. Newly obtained sequences in this study are in labelled as study isolates. Analysis was done using 1000 bootstrap replicates. The bootstrap support value for each clade is indicated on the nodes.
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In India, it was first reported by Poornima et This is primarily because DNA based methods al (2016) from guava orchards of Ayakudi are rapid and reliable when compared to villages of Dindigul district of Tamil Nadu. morphological or biochemical methods Various authors reported M. enterolobii from (Powers et al., 2005). various locations of guava orchards in the states of Pernambuco and Bahia (Carneiro et The most popularly used DNA based methods al., 2001), Rio de Janeiro (Lima et al., 2003), are mitochondrial DNA (mtDNA), sequence Ceará (Torres et al., 2005), São Paulo characterized amplified region markers (Almeida et al., 2006), Paraná (Carneiro et al., (SCAR), amplified fragment length 2006), Piauí (Silva et al., 2006) and Espírito polymorphisms (AFLP), random amplified Santo (Lima et al., 2007). However, earlier it polymorphism DNA (RAPD), ribosomal DNA was misidentified as M. incognita (Fargette et (rDNA), restriction fragment length al., 1994 and Brito et al., 2004). The present polymorphisms (RFLPs), microsatellite DNA study used a different combination (Perineal (satDNA), microarrays and real time PCR pattern, morphometrics and DNA bases) tools (qPCR). In the present study, M. enterolobii for identification of M. enterolobii in guava from guava was identified through PCR using orchards of Tamil Nadu. Analysis of perineal ITS region of Nematodes. patterns requires considerable skill because of significant intraspecific variations (Carneiro et The method for identification of root knot al., 1996). In this study, minor variations were nematode species using PCR was first observed in perineal pattern of three district reported by Harris et al. (1990), who population isolates when compared with amplified the mt DNA from single second previously published M. enterolobii perineal stage juvenile and this method was further patterns, which could due to influence of developed by Powers & Harris (1993) who varietal (of the host) or edaphic factors. designed primers for amplifying the intervening region between the mt DNA gene Molecular characterization of M. coding for cytochrome oxidase subunit II and enterolobii the 16S rRNA primers and used them to identify five major Meloidogyne species viz., Root knot nematode, M. enterolobii was M. incognita , M. javanica, M. arenaria, M. confirmed by DNA based methods. The haplaand M. chitwoodi. primers (TW81 and AB28) led to positive amplifications and sequencing for all the Regions of the nuclear genomes of 18Sand isolates. The amplicon size were 28S rDNA have been used for characterization approximately 600 bp of ITS region of of root knot nematode species. Ribosomal Meloidogyne spp. (Fig. 3). Amplified PCR DNA has been very informative regions for products were purified and partially sequenced diagnostic and phylogenetic studies of plant at Chromous Biotech PVT Ltd. Bangalore, parasitic nematodes (Blok, 2005). India. The sequences were deposited in BLAST and Gene accession numbers viz., Kiewnick et al. (2009) reported that, no Dindigul populations (MK940246 & differences were found at DNA level between MK955348), Coimbatore populations two species M. enterolobii and M. (MK955350, MN006626) and Mayaguensis, and they concluded M. Thiruvannamalai populations (MN381161 and enterolobii as synonyms of M. mayaguensis MN381109) were obtained. Various molecular and M. mayaguensis asjunior synonym of M. approaches have been designed for enterolobii. identification of root knot nematode species. 1994
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Phylogenetic analysis Brito, J., Powers, T.O., Mullin, P.G., Inserra, R.N and Dickson, D.W. 2004. DNA sequences of the internal transcribed Morphological and molecular spacer (ITS) have been useful for characterization of Meloidogyne identification of nematode species (Zijlstraet mayaguensis isolates from Florida. al., 1997). In the present study, an Journal ofNematology. 36:232-240. approximately 600 bp DNA fragment of Brito, J.A., Stanley, J.D., Kaur, R., Cetintas, internal transcribed spacer (18S-28S) of R., Di Vito, M., Thies, J.A. & Dickson, M. enterolobii nematode population were D.W. 2007. Effects of the Mi-1, N and amplified and partially sequenced. Sequences Tabasco genes on infection and were aligned with other Meloidogyne species reproduction of Meloidogyne obtained from GenBank database and the mayaguensis on tomato and pepper phylogenetic tree was constructed. genotypes. Journal of Nematology39, 327-332. The Maximum Likelihood tree (Fig 4)was Brito, J.A., Kaur, R., Cetintas, R., Stanley, highly resolved and showed that in the present J.D., Mendes, M.L., Powers, T.O. & study, populations of M. enterolobii formed a Dickson, D.W. 2010. Meloidogyne spp. clade together with sequences other M. infecting ornamental plants in Florida. enterolobii (MK975452 and KF418369) Nematropica40, 87- 103. populations retrieved from GenBank database. Blok, V. 2005. Achievements in and future Separate clades were formed for other species prospects for molecular diagnostics of of Meloidogynewith high bootstrap support plant-parasitic nematodes. (Tamura et al., 2011). Canadian.Journal Plant Pathology.27: 176-185. In summary, results of this study demonstrated Blok, V.C., Phillips, M.S and Fargette, M. clearly the presence of M. enterolobii in all the 1997. Comparison of sequences from six isolates collected from three districts of the ribosomal DNA intergenic region of guava growing orchards of Tamil Nadu, Meloidogyne mayaguensis and other identified based on morphological and major tropical root knot nematodes. molecular methods. Journal of Nematology. 29:16-22. Cobb, N.A. 1918. Estimating the nematode Acknowledgement population of soil. U.S. Dep. Agr. Bur. plant Ind. Agr. tech. Cir. 1:1-48. Authors acknowledge to Department of Cenis, J.L. 1993. Identification of four major Nematology and Plant Pathology, Tamil Nadu Meloidogyne spp. by random amplified Agricultural University, Coimbatore, 641003, polymorphic DNA (RAPDPCR). Tamil Nadu, India. Phytopathology. 83: 76-78. Carneiro, R. M. D. G., Carneiro, R. G., References Abrantes, I. M. O., Santos, M. S. N., and Almeida, M. R. A. 1996. Almeida, E.J., Soares, P.L.M., Santos, J.M. & Meloidogyneparanaensis n. sp. Martins, A.B.G. 2006. Estudo da (Nemata: Meloidogynidae), a root-knot resistência de araçás (Psidium spp.) nematode parasitizing coffee in Brazil. (Mirtaceae) a Meloidogyne mayaguensis J. Nematol. 28:177-189. em casa de vegetação. Nematologia Carneiro, R.M.D.G., Moreira, W.A., Almeida, Brasileira 30:118-119. R.M.A. & Gomes, A.C.M.M. 2001.
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How to cite this article:
Suresh, P., K. Poornima, P. Kalaiarasan, S. Nakkeeran and Vijayakumar, R. M. 2019. Characterization of Guava Root Knot Nematode, Meloidogyne enterolobii Occurring in Tamil Nadu. Int.J.Curr.Microbiol.App.Sci. 8(09): 1987-1998. doi: https://doi.org/10.20546/ijcmas.2019.809.230
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