Morphological, enzymatic and molecular characterization of root-knot nematodes parasitizing vegetable crops

Caracterização morfológica, enzimática e molecular de nematoide das galhas, parasitas de hortaliças

ABSTRACT

Species of the genus Meloidogyne are limiting factors in vegetable crop production. Studies in Brazil about the occurrence of root-knot nematodes in areas of vegetable crop growth have been conducted without using advanced techniques. Using modern techniques, such as biochemical and molecular methods, improves the accuracy of Meloidogyne species identification. The present study characterized species of Meloidogyne in 36 samples associated with vegetable crops using isoenzyme electrophoresis, SCAR markers, and morphological markers, in addition to validating SCAR markers for accurate species identification. The species M. incognita, M. javanica, M. hapla, M. morocciensis, and M. arenaria were identified, with the first two being the most frequent. Here, the species M. arenaria parasitizing scarlet eggplant and M. morocciensis parasitizing pumpkin and cabbage are reported in Brazil for the first time. Esterase electrophoresis efficiently separated the species of Meloidogyne found in vegetable crops; however, SCAR markers were only effective for the identification of M. incognita, M. javanica, and M. hapla, since the primer pair Far/Rar yielded no amplification product to confirm the identity of M. arenaria. The species M.arenaria and M. morocciensis could not be distinguished by the female perineal patterns. Based on the present results, new primers should be designed for the identification of M. arenaria and M. morocciensis.

Keywords:
Meloidogyne; diagnosis; molecular biology; vegetable crops

RESUMO

Espécies do gênero Meloidogyne constituem um dos fatores limitantes à produção de olerícolas. Estudos têm sido realizados no Brasil a respeito da ocorrência de espécies de nematoide de galhas em áreas de cultivo de olerícolas sem o uso de técnica avançada. O uso de técnicas modernas, tais como, os métodos bioquímicos e moleculares aumentam a segurança na identificação das espécies de Meloidogyne. Neste trabalho foram caracterizadas espécies de Meloidogyne associadas a olerícolas em um total de 36 amostras por meio de eletroforese de isoenzimas, marcadores SCAR e morfológico. Além disso, foram validados marcadores SCAR para identificação acurada dessas espécies. Desta forma, foram identificadas as espécies M. incognita, M. javanica, M. hapla, M. morocciensis e M. arenaria, sendo as duas primeiras espécies mais frequentes. Foram ainda relatadas, pela primeira vez no Brasil, as espécies: M. arenaria parasitando plantas de jiló e M. morocciensis parasitando plantas de abóbora e repolho. A eletroforese de esterase foi eficiente em separar todas as espécies de Meloidogyne encontradas em olerícolas. Entretanto, os marcadores SCAR foram eficientes, somente, na identificação de M. incognita, M. javanica e M. hapla, no entanto, os primers Far/Rar não promoveram amplificação para confirmação da identidade de M. arenaria. Na avaliação da configuração perineal de fêmeas, não foi possível distinguir as espécies M. arenaria e M. morocciensis. Com base nos resultados, novos primers devem ser desenvolvidos para identificação de M. arenaria e M. morocciensis.

Palavras chave:
Meloidogyne; diagnose; biologia molecular; hortaliças

Plant parasitic nematodes are the major pest problem in vegetable crops in the tropics (Carneiro et al., 2008b). In certain production areas, root-knot nematodes are responsible for an approximate 30% yield reduction in vegetable crops (Anwar et al., 2009). Vegetable productivity reductions are directly related to preplant infestation levels in the soil; as soil infestation increases, the amount of damage and yield loss also increases (Noling, 2012). Meloidogyne is the most important genus in Brazil due to its wide distribution throughout different vegetable production regions, polyphagia, and physiological variability among populations of the same species (Moens et al., 2009).

There exist three techniques for Meloidogyne identification, representing the evolution of knowledge regarding this nematode species over time: perineal pattern, electrophoresis of isoenzymes, and sequence-characterized amplified region (SCAR). Firstly, in 1949, Chitwood described the Meloidogyne genus and subsequently developed a perineal pattern method that was able to distinguish between five species of Meloidogyne. During 1950−70, the descriptions were improved (Taylor & Sasser, 1978). Secondly, application of electrophoresis to Meloidogyne species identification was well established by Esbenshade & Triantaphyllou (1990). Thirdly, studies by Zijlstra et al. (2000); Randig et al. (2002); Meng et al. (2004) and Tigano et al. (2010) using SCAR markers were widely accepted by plant-parasitic nematode researchers.

Identifying Meloidogyne species based only on morphological characteristics such as the perineal pattern in females is not reliable, since the analysis may not reflect the species diversity. For instance, Meloidogyne incognita in coffee was characterized for 22 years as M. incognita race 5 or biotype IAPAR, which only occurred in Brazil; however, it was defined as a novel species by electrophoresis (Carneiro et al., 1996). The electrophoresis of isoenzymes is more precise and permits the identification of species in a mixture and characterization of atypical populations (Carneiro et al., 2016). Nevertheless, identification using this technique is limited to the stage of adult females, but females are not always suitable for analysis due to the state of root degradation (Salgado et al., 2015). SCAR markers, however, represent a fast and accurate tool with which to determine the identity of Meloidogyne species, in addition to allowing the detection of species in a mixture and making it possible to identify species based on eggs and juveniles, which eliminates the problem of inadequate females (Salgado et al., 2015). Yet, only nine Meloidogyne species detected in Brazil can be identified by SCAR markers (Carneiro et al., 2016).

The main Meloidogyne species parasitizing Brazilian vegetables are Meloidogyne javanica, M. incognita, M. arenaria, and M. hapla. Another species of great importance, M. enterolobii, has also been detected in vegetables (Carneiro et al., 2006; Almeida et al., 2008). Despite the importance of the correct identification of Meloidogyne species, few studies in Brazil have characterized these species based on esterase and SCAR markers. Using electrophoresis analysis, M. enterolobii was found in the states of Mato Grosso, Ceará, São Paulo, and eastern Minas Gerais (Carneiro et al., 2006; Oliveira et al., 2007; Almeida et al., 2008; Silva et al., 2016); M. incognita, M. javanica, and M. arenaria were found in eastern Minas Gerais (Oliveira et al., 2007); and M. ethiopia was reported in the Federal District (Carneiro et al., 2005). Based on electrophoresis and SCAR markers, different species were identified in Brazil in association with potatoes, such as M. javanica, M. incognita, M. arenaria, and M. ethiopica (Medina et al., 2017). Using only the perineal pattern technique, M. incognita, M. javanica, M. arenaria, and M. hapla were detected in several potato-growing states in Brazil (Charchar, 1997), and M. incognita and M. javanica were found in vegetable crops in Maranhão (Silva, 1991).

The combined use of enzymatic, molecular, and morphological methods better characterize the diversity of Meloidogyne species in vegetables with higher identification reliability. Breeders and agribusiness consultants benefit from these studies with respect to the selection of genotypes for research and the recommendation of cultivars specific to the species of nematode. Familiarization and validation of biomolecular protocols for Meloidogyne tropic species are also important.

Thus, the objectives of the present study were to: 1) evaluate the diversity of Meloidogyne species associated with vegetables in various states in Brazil using morphological, enzymatic, and molecular characterization; and 2) validate the SCAR markers for the accurate identification of these species, with a view to such markers being used in diagnostic laboratories in the future.

MATERIAL AND METHODS

Collecting and obtaining females of Meloidogyne spp.

Thirty-six populations of Meloidogyne were obtained from vegetable-producing areas in seven different states in Brazil (Table 1). Five sub-samples were collected composing one sample of soil and roots removed from the rhizosphere of plants with root-knot symptoms at approximately 20-cm depth. The samples were placed in plastic bags, stored in styrofoam boxes with identification labels, and subsequently processed.

Table 1
Phenotype of esterase and species names of Meloidogyne associated to vegetable with galls in several states, Brazil. Lavras, UFLA, 2015.

Population Code	Nematode species	Geographical origin	Host plant	Phenotype of esterase
1	M. incognita	Viçosa-MG	Cucurbita pepo	I1
2	M. arenaria	Cajuri-MG	Solanum aethiopicum	A2
3	M. morocciensis	Cajuri-MG	Cucurbita pepo	A3
4	M. javanica and M. incognita	Cajuri-MG	Solanum lycopersicum	J3 and I1
5	M. javanica	Cajuri-MG	Solanum aethiopicum	J3
6	M. incognita	Cajuri-MG	Cucurbita pepo	I2
7	M. incognita	Santana da Vargem-MG	Lactuca sativa	I2
8	M. arenaria and M. incognita	Santana da Vargem-MG	Lactuca sativa	A2 and I1
9	M. incognita	Santana da Vargem-MG	Abelmoschus esculentus	I1
10	M. hapla	Pouso Alegre-MG	Fragaria vesca	H1
11	M. javanica	Lavras-MG	Solanum tuberosum	J3
12	M. javanica	Lavras-MG	Lactuca sativa	J3
13	M. incognita	Ijaci-MG	Cucurbita pepo	I1
14	M. javanica and M. hapla	Rio Paranaíba-MG	Daucus carota	J3 and H1
15	M. javanica and M. incognita	Rio Paranaíba-MG	Solanum tuberosum	J3 and I1
16	M. incognita	Rio Paranaíba-MG	Daucus carota	I1
17	M. javanica and M. incognita	Rio Paranaíba-MG	Daucus carota	J3 and I2
18	M. incognita	Muzambinho-MG	Cucumis sativus	I2
19	M. incognita	Ibertioga-MG	Arracacia xanthorrhiza	I2
20	M. javanica	Piedade-SP	Beta vulgaris	J3
21	M. javanica	Piedade-SP	Solanum lycopersicum	J3
22	M. hapla	Piedade-SP	Solanum lycopersicum	H1
23	M. incognita	Piedade-SP	Lactuca sativa	I2
24	M. incognita, M. javanica and M. arenaria	Piedade-SP	Cichorium endivia	I1, J3 and A2
25	M. incognita	Holambra-SP	Solanum lycopersicum	I1
26	M. javanica and M. incognita	Casa Nova-Ba	Cucumis melo	J3 and I1
27	M. morocciensis	Mucugê-Ba	Brassica oleracea	A3
28	M. incognita	Piracanjuba-GO	Abelmoschus esculentus	I1
29	M. incognita	Burite Alegre-GO	Solanum lycopersicum	I2
30	M. javanica	Caldas Novas-GO	Abelmoschus esculentus	J3
31	M. incognita	Bom Jesus-GO	Solanum aethiopicum	I1
32	M. javanica	Morrinhos-GO	Abelmoschus esculentus	J3
33	M. javanica	Petrolina-PE	Solanum lycopersicum	J3
34	M. javanica and M. incognita	Petrolina-PE	Beta vulgaris L.	J3 and I1
35	M. javanica	Venda Nova do Imigrante-ES	Solanum lycopersicum	J3
36	M. arenaria	Pelotas-RS	Solanum tuberosum	A3

Nematodes were extracted from the roots containing galls. Roots were rinsed in standing water and dissected with tweezers and fine scalpels under a stereoscopic microscope to release the milky-white females. The females were used for subsequent evaluation of the perineal pattern and isoenzyme electrophoresis.

Multiplication of Meloidogyne spp. populations

Samples with roots in an advanced rotting state and not containing appropriate females for morphological and electrophoretic studies had the roots washed in standing water. Subsequently, the samples were cut into 0.5-cm pieces, and the eggs extracted using the process described by Hussey & Barker (1973). Tomato seedlings (cv. Santa Clara) were inoculated with the eggs in a greenhouse for nematode multiplication for approximately two months, when it was possible to obtain appropriate females for the study.

Morphological studies of Meloidogyne spp. populations

Ten milky-white females removed from infected roots were immediately transferred to one drop of 45% lactic acid. The perineal area of each female was cut, cleaned, and mounted on slides with glycerol for identification using a light microscope.

Isozyme characterization of Meloidogyne spp. populations

Adult females of milky-white color in early oviposition were taken either from newly collected field samples or tomato roots resulting from greenhouse multiplication for 60 days. The females were transferred to 0.2-mL microfuge tubes containing 10 µL protein extraction buffer, according to Davis (1964), and were subsequently smashed with a rounded-end glass rod in an ice block to prevent protein denaturation. Vertical polyacrylamide gel electrophoresis was used with a bis-acrylamide concentration of 8% for the stacking gel and 4% for the running gel. When electrophoresis was not performed on the same day, the females were kept in the freezer at a temperature of approximately-10^°C.

For electrophoresis, nine females were used for each population, e.g., one female per well. The protein extract from M. javanica was placed in the first, sixth, and final well of each gel as a standard for comparison with the phenotypes found. Electrophoresis was performed in a refrigerator at approximately 4°C, with a voltage of 80 V through the stacking gel for 15 minutes and 200 V through the running gel for 30 minutes. Migration was monitored by means of displacement of the frontline of bromophenol blue, and electrophoresis was stopped when this line was 1 cm from the bottom of the gel. After stopping the electrophoretic run, the gel was removed from the plate. The running gel was immersed in a developing solution prepared immediately prior to use, to study esterase isozyme (EST) pattern. The protein bands were compared with the positions presented by the migration of the M. javanica esterase pattern, as reported in the literature (Esbenshade & Triantaphyllou 1990; Carneiro et al., 2008a).

DNA extraction

The extraction of genomic DNA was performed using the protocol described by Holterman et al. (2006), with slight modifications. To obtain nematodes, the roots were cut into 0.5-cm pieces, from which eggs were extracted using the procedure described by Hussey & Barker (1973). The eggs were placed in a hatching chamber, and 10 second-stage juveniles (J2) were transferred to PCR microtubes containing 50 µL HLB buffer [0.2 M NaCl, 0.2 M Tris-HCl (pH= 8.0), 1%(v/v) β-mercaptoethanol, and 800 µL/mL proteinase K]. The samples were centrifuged at 14,000 rpm for one minute, incubated at 65°C for 2 hours and 99°C for 5 minutes, and subsequently stored at -20°C for later use in PCR reactions.

Identification of Meloidogyne species by SCAR markers

Based on studies of perineal pattern and esterase phenotypes, a prior identification was carried out and confirmed using SCAR markers. The SCAR markers used to identify the species of Meloidogyne are listed in Table 2.

Table 2
Sequences of SCAR markers used for identifying species of Meloidogyne. Lavras, UFLA, 2015.

SCAR Primers	Sequence (5’→ 3’)	Size of the SCAR (bp)	Identified species	References
FhN RhN	GCCTTCTTTGGATTCCTCTCA GGCTCATCCTTGCTGTAAAT	420	M. hapla	Zijlstra (2000)
Far Rar	TCGGCGATAGAGGTAAATGAC TCGGCGATAGAGGTAAATGAC	420	M. arenaria	Zijlstra et al. (2000)
Fjav Rjav	GGTGCGCGATTGAACTGAGC CAGGCCCTTCAGTGGAACTATAC	670	M. javanica	Zijlstra et al. (2000)
inc-K14-F inc-K14-R	GGGATGTGTAAATGCTCCTG CCCGCTACACCCTCAACTTC	399	M. incognita	Randig et al. (2002)

Amplification reactions using the SCAR markers were performed in a final volume of 25 µL, containing 1 µL DNA, 0.7 µL each primer (10 μM), 0.5 µL acetylated BSA (10 mg/mL), 12.5 µL GoTaq Colorless PCR Master Mix (Promega, Madison, USA), and 9.6 µL nuclease-free water. PCR reactions were performed using a My Cycler ^TMthermal cycler (BIO-RAD). The amplification conditions for each primer set are described in Table 3. All primers were synthesized by Sigma-Aldrich (Madrid, Spain). The PCR products were subjected to agarose gel electrophoresis at 0.7% and stained with GelRed (Biotium^®) to visualize the bands. The length of each amplified fragment was compared with the Axygen 1 kb DNA ladder.

Table 3
Amplification conditions used for different primers in identification of Meloidogyne species. Lavras, UFLA, 2015.

Primers	35 cycles
FhN/RhN	2 min/ 94oC	30 s/94oC	30 s/58o C	1 min/72oC	4oC /∞
Far/Rar	2 min/ 94oC	30 s/94oC	30 s/61o C	1 min/72oC	4oC /∞
Fjav/Rjav	2 min/ 94oC	30 s/94oC	30 s/64° C	1 min/72oC	4oC /∞
inc-K14-F/inc-K14-R	5 min/ 940C	30 s/94oC	45 s/55oC	1 min/70oC	8 min/ 70oC	4oC /∞

RESULTS AND DISCUSSION

In the present study, analysis of the diversity of Meloidogyne species in 36 soil samples and vegetable roots with galls collected from seven different states in Brazil yielded six different species: M. incognita, M. javanica, M. hapla, M. arenaria, and M. morocciensis (Table 1). The most frequent species were M. incognita (55.5%) and M. javanica (44.4%), and the least frequent were M. arenaria (11.1%), M. hapla (8.3%), and M. morocciensis (5.5%). Mixed populations occurred in 22% samples. The predominance of the M. incognita and M. javanica species in vegetable crops and the lower frequency of M. arenaria and M. hapla found in the present study are in accordance with reports by Charchar (1997), Silva (1991), Oliveira et al. (2007), and Rosa et al. (2013); however, only the latter two references used electrophoretic pattern of esterase for species identification. Nevertheless, to date, there have been no reports of M. arenaria parasitizing scarlet eggplant (Solanum aethiopicum) or M. morocciensis parasitizing pumpkin (Cucurbita pepo) and cabbage (Brassica oleracea) in Brazil.

The species found in the present study showed the following characteristics: M. incognita had a high trapezoidal dorsal arch and striae varying from fine to coarse; M. javanica had a distinct perineal pattern with incisures on the lateral lines, interrupting the streaks of ventral and dorsal fields, where few or no striae crossed the lateral lines of the perineal pattern; M. hapla had smooth striae, a rounded dorsal arch, and visible points near the end of the tail; M. arenaria and M. morocciensis had a similar perineal pattern, becoming impossible to distinguish, with a low rounded dorsal arch, coarse striae, and some striae bent toward the vulva. All perineal patterns were similar to those already described by Jepson (1987) and Carneiro et al. (2008a).

Although perineal patterns have long been used for the identification of Meloidogyne species, this method has shown inconsistent results, besides being subjective and prone to many mistakes (Carneiro et al., 2004). For instance, M. arenaria and M. morocciensis cannot be distinguished by perineal patterns, which has also been corroborated by Conceição et al. (2012). Perineal patterns of M. incognita described here were also similar to other species such as M. paranaensis, M. enterolobii (Syn. M. mayaguensis), and M. izalcoensis.

In the present study, the electrophoretic pattern of esterase was sufficient to distinguish Meloidogyne species, including M. arenaria and M. morocciensis, leaving no doubt of species identity. Of the 36 Meloidogyne populations assessed, we found six phenotypes in electrophoretic analysis using esterase (Table 1; Figure 1). The phenotypes were J3, characteristic of M. javanica; I1 and I2, characteristic of M. incognita; H1, characteristic of M. hapla; A2, characteristic of M. arenaria; and A3, characteristic of M. morocciensis. The phenotype I1 (66.7%) of M. incognita was more frequent than I2 (33.3%) of the same species. Other studies have used isozyme electrophoresis to confirm identities by perineal patterns (Oliveira et al., 2007; Carneiro et al., 2008a; Barros et al., 2011).

Figure 1
Phenotypes of esterase of Meloidogyne spp. associated with vegetable crops. A: phenotype I1 of M. incognita; B: phenotype I2 of M. incognita; C: phenotype A2 of M. arenaria; D: phenotype A3 of M. morocciensis; E: phenotype H1 of M. hapla; F: phenotype J3 of M. javanica. Mj: phenotype of M. javanica used as comparing patterns. Lavras, UFLA, 2015.

The pairs of SCAR markers usually employed in the diagnosis of Meloidogyne species were more efficient in distinguishing the species M. incognita, M. javanica, and M. hapla associated with vegetable crops (Figure 2); however, the primer pair (Far/Rar) indicated for the identification of M. arenaria was inefficient, since no PCR product was detected even following optimization of the annealing temperature. In accordance, Carneiro et al. (2008a) found no amplification of DNA fragments of three populations of the M. arenaria phenotype A2 using the same primer pair. Therefore, the development of a novel pair of species-specific primers for M. arenaria and M. morocciensis is necessary.

Figure 2
Specific amplification by PCR from DNA of Meloidogyne spp. associated with vegetables crops: (A) inc-K14-F/inc-K14-R, (B) Fjav/Rjav and (C) FhN/RhN. M: 1 kb plus ladder; 1-36 populations of Meloidogyne; NC: negative control; PC: positive control. Lavras, UFLA, 2015.

Several field samples showed mixed Meloidogyne populations (Table 1). Since DNA was extracted from 10 J2 samples, some could have predominated, such as sample 8, in which M. arenaria prevailed over M. incognita; thus, the sample DNA was not amplified using the inc-K14-F/inc-K14-R primer pair.

Root samples sometimes arrive already degraded to the laboratory, precluding withdrawal of females or even eggs; thus, DNA extraction from J2 may facilitate diagnosis in laboratories, since J2 may be found in the soil.

Isozyme electrophoresis in conjunction with SCAR markers was efficient in diagnosing Meloidogyne species that parasitize vegetable crops, expanding the pool of Meloidogyne species in different states in Brazil. Despite SCAR markers being efficient, only nine species could be identified in Brazil by this method, whereas there is a pattern of esterase for all species detected in Brazil, which makes isoenzyme electrophoresis more advantageous (Carneiro et al., 2016). In the present study, we verified that the primer pair Far/Rar does not identify M. arenaria, and there is no primer described for M. morocciensis; however, the inc-K14-F/inc-K14-R, Fjav/Fjav, and FhN/RhN primer pairs were validated to characterize M. incognita, M. javanica, and M. hapla, respectively. Molecular, enzymatic, and morphological techniques must be used in combination to provide reliable diagnosis.

Our data allowed us to access the diversity of Meloidogyne species associated with vegetables crops, and to verify that a plant may be infected with more than one species simultaneously. Here, we report, for the first time in Brazil, the presence of M. arenaria parasitizing scarlet eggplant and M. morocciensis parasitizing pumpkin and cabbage. The present findings will contribute to future studies regarding genetic improvement and control methods.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the Brazilian financial supports provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), through process n° 457917/2014-8, and other supports, as well as Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Publication Dates

• Publication in this collection
  Oct-Dec 2018

History

• Received
  04 Sept 2017
• Accepted
  10 Oct 2018