Intra-racial genetic variation of Ditylenchus dipsaci isolated from garlic in Ontario as revealed by random amplified polymorphic DNA analysis

Abstract

The stem and bulb nematode (Ditylenchus dipsaci) was found in garlic from across Ontario in a survey conducted in 2011. Nematodes from the survey were extracted from garlic samples and the identification of Ditylenchus species was established using morphological and molecular methods. The genetic variation of 90 populations collected from different regions in the province was studied using the random amplified polymorphic DNA (RAPD) method. One population from onion and one population of D. destructor were included for comparison. Simple matching coefficiency and hierarchical cluster analysis were performed on the polymorphisms to demonstrate the intra-racial, intra-species and interspecies relationships. Genetic variation was detected in the D. dipsaci populations isolated from garlic in Ontario. All the D. dipsaci populations were separated into two distinct clusters: a small cluster consisting of 19 populations from counties in the southern part of the province, and a large cluster of 71 populations mostly from the eastern region but which also included a few populations from the southern western and northern regions of the province. The average similarity coefficiency among populations of D. dipsaci isolated from garlic was 82%. The grouping of the two distinct clusters seems to suggest that there were two separate introductions of D. dipsaci in Ontario. This is the first report of intra-racial genetic variations of D. dipsaci.

Résumé

L'anguillule des tiges et des bulbes (Ditylenchus dipsaci) a été trouvée dans de l'ail cultivé partout en Ontario au cours d'une étude menée en 2011. Les nématodes trouvés provenaient d’échantillons d'ail et l'espèce Ditylenchus a été identifiée au moyen de techniques morphologiques et moléculaires. La variation génétique de 90 populations collectées dans différentes régions de la province a été étudiée par amplification aléatoire de l'ADN polymorphe. Une population d'oignons et une de D. destructor ont été incluses à des fins de comparaison. Le coefficient de simple appariement et la classification hiérarchique ont été appliqués au polymorphisme pour démontrer les relations intraraces, intraespèces et interespèces. La variation génétique a été détectée chez les populations de D. dipsaci isolées de l'ail provenant d'Ontario. Toutes les populations de D. dipsaci ont été divisées en deux familles distinctes : une petite, consistant de 19 populations provenant de comtés de la portion sud de la province, et une grande, comportant 71 populations provenant principalement des régions est, mais également du sud-ouest et du nord de la province. L'indice de similitude moyen chez les populations de D. dipsaci isolées des échantillons d'ail était de 82 %. Le regroupement des deux familles distinctes semble suggérer qu'il y a eu deux vagues d'introduction de D. dipsaci en Ontario. Il s'agit de la première mention de variation génétique intraraciale chez D. dipsaci.

Keywords:

• Ditylenchus dipsaci
• genetic variation
• intra-racial
• nematode
• RAPD

Mots clés:

• amplification aléatoire de l'ADN polymorphe
• Ditylenchus dipsaci
• intraracial
• nématode
• variation génétique

Introduction

The stem and bulb nematode, Ditylenchus dipsaci (Kuhn, 1857) (Filipjev, 1936), is one of the most devastating plant parasitic nematodes, and it can infest over 500 plant species, many of which are important crops. It is known to have more than 30 biological races defined by their host preference (Eriksson, 1974; Sturhan & Brzeski, 1991). The populations infesting garlic and onion are commonly considered ‘the onion race’. Although some races are polyphagous, no races other than ‘the onion race’ are known to be able to establish on garlic. The pest is cosmopolitan and anhydrobiotic, the latter is an uncommon trait in nematodes (Barrett, 1991). The species was not fully accepted until 1945 (Thorne, 1945), and it has had about 25 synonymies and its taxonomic position has been changed a few times (Siddiqi, 2000). All these attributes make this pest difficult to identify and to manage. It is regulated internationally in many crops but not in garlic in North America. Garlic growers in Ontario, Canada, and several northeastern US states have seen crop losses as high as 80–90% in sections of fields infested with D. dipsaci (Abawi & Moktan, 2010; Thomas, 2011). The loss of entire fields of garlic due to D. dipsaci infestation has been reported by farmers in Ontario (Celetti, 2011). It is suspected that the quick spread of the pest was largely due to the exchange and trade of infested garlic cloves used for seeds.

Ditylenchus dipsaci is believed to be native to Europe, and was probably accidentally introduced to Canada by the early European settlers (Gussow, 1931). It has been reported in most provinces in Canada including Alberta (Hawn, 1973), British Columbia (Bocher, 1960; Vrain, 1987; Elmhirst & Joshi, 1997), Ontario (Mountain, 1957), Prince Edward Island (Kimpinski, 1985), Quebec (Lacroix et al., 1997), and Saskatchewan (Creelman, 1963). In Ontario, prior to this recent outbreak (Sabourin et al., 2006; Yu et al., 2010), D. dipsaci had been found in a few isolated fields in onion. It was first reported in onions from the Leamington Marsh, Essex County (Mountain, 1957), followed by findings in the Erieau Marsh, Kent County (Johnson & Kayler, 1972) and in Cookstown, Simcoe County in 1975 (Fushtey & Kelly, 1975). In a recent survey carried out by the Garlic Growers Association of Ontario in 2011, D. dipsaci was found to be widespread in garlic from across the province (Hughes et al., 2012, in press). There are approximately 500 hectares of garlic grown in Ontario. Of the garlic tested during the survey, 73% were found to have stem and bulb nematodes. An internationally quarantined pest, D. destructor (Thorne, 1945), the potato rot nematode, was found unexpectedly in this survey in a 0.5 hectare garlic field of eastern Ontario (Yu et al., 2012).

It has long been known that natural selection forces intraspecific variation and intraspecific variation is a necessary process for evolution and speciation. Intraspecific variation in the pathogenicity of plant parasitic nematodes has long been recognized, i.e., D. dipaci, Globodera rostochinesis and G. pallida, Heterodera glycines, Meloidogyne spp. (Dropkin, 1988). Different terms such as race, biotype, pathotype and strain have been used to denote these phenotypic variances. With the rapid development of molecular techniques such as random amplified polymorphic DNA (RAPD) analysis, restriction fragment length polymorphism (RFLP) analysis, and the analysis of expressed sequence tags (EST), the underlying genetics of these intraspecific variances are gradually being recognized for some of the most important plant parasitic nematodes such as H. glycines (Radice et al., 1988), G. rostochiensis and G. pallida (Folkertsma et al., 1994), and Meloidogyne spp. (Castagnone-Sereno et al., 1994). Based on this genetic knowledge, resistance genes have been discovered and resistant cultivars have been developed for the management for some of the pests (Dale & de Scurrah, 1998; Shannon et al., 2004; Williamson et al., 1994).

Ditylenchus dipsaci, probably the first nematode pest to be recognized to have different races (Seinhorst, 1956), is now known to consist of over 30 races. Different races of D. dipsaci have different host ranges and generally one race will not infest the hosts of another; however, some races appear to be polyphagous. The onion race is among the latter (Webster, 1967). Knowledge on the underlying molecular and genetic differences of the nematode has gradually been accumulated at the race level. Much effort had been focused on the giant race, a race infesting faba bean (Vicia faba L.), which has a greater body size than other races (normal races) (Esquibet et al., 1998, 2003; Subbotin et al. 2003, 2005). The giant race has been raised to the species level, as D. gigas (Vovlas et al., 2011). Little is known regarding intraspecific variations in D. dipsaci below the race level (intra-racial).

It is important to understand the genetic basis of a pest in order to develop successful management strategies. The objective of this study was to use RAPD fingerprinting to assess the intra-racial genetic variation within populations of D. dipsaci isolated from garlic in Ontario, relative to a population from onion and a closely related species D. destructor. Parts of the results have been published previously (Qiao et al., 2012).

Table 1. Nematode samples collected from 26 counties in Ontario, Canada

Code	County/District	Cultivar	Species	Code	County/District	Cultivar	Species
A-01	Elgin	Music	D. dipsaci	D-74	Wellington	Music	D. dipsaci
A-03	Essex	unknown	D. dipsaci	D-75	Wellington	Music	D. dipsaci
A-04	Middlesex	Music	D. dipsaci	D-76	Wellington	Music	D. dipsaci
A-05	Perth	Music	D. dipsaci	D-77	Wellington	Music	D. dipsaci
A-06	Bruce	Music	D. dipsaci	D-78	Elgin	Music	D. dipsaci
A-07	Wellington	Music	D. dipsaci	D-79	Elgin	Music	D. dipsaci
A-08	Municipality of York	Music	D. dipsaci	D-80	Elgin	Music	D. dipsaci
A-09	Huron	Music	D. dipsaci	D-82	Temiskaming	Music	D. dipsaci
A-10	Brant	Music	D. dipsaci	D-83	Temiskaming	Music	D. dipsaci
A-11	Bruce	Music	D. dipsaci	D-121	Elgin	Music	D. dipsaci
A-112	Bruce		D. dipsaci	E-102	Lanark	Georgia Fire	D. dipsaci
A-12	Bruce	Music	D. dipsaci	E-103	Lanark	French Rocambole	D. dipsaci
A-13	Brant	Music	D. dipsaci	E-104	Carleton/Ottawa	Native Creole	D. dipsaci
A-14	Middlesex	Music	D. dipsaci	E-105	Lanark	Red Russian	D. dipsaci
A-15	Norfolk	Music	D. dipsaci	E-106	Frontenac	French Rocambole	D. dipsaci
A-16	Essex	Ricardo (porcelain)	D. dipsaci	E-107	Lanark	French Rocambole	D. dipsaci
A-17	Perth	Music	D. dipsaci	F-92	Manitoulin	Siberian	D. dipsaci
A-18	Peel Region	Music	D. dipsaci	F-108	Carleton/Ottawa	McEwen	D. dipsaci
A-20	Perth	Mexican Purple	D. dipsaci	F-109	Carleton/Ottawa	Music	D. dipsaci
A-21	Perth	Rocambole	D. dipsaci	F-110	S. Dundas/ Stormont	Music	D. dipsaci
A-22	Perth	Music	D. dipsaci	F-124	Niagara	Music	D. dipsaci
A-23	Perth	Spanish Roja	D. dipsaci	EB	Halton		D. dipsaci
B-1	Brant	Music	D. dipsaci	G-125	Renfrew	Siberian	D. dipsaci
B-2	Carleton/Ottawa	Yugoslavian	D. dipsaci	G-127	Renfrew	German	D. dipsaci
B-3	Carleton/Ottawa	Carpathian	D. dipsaci	G-128	Grey	Music	D. dipsaci
B-4	Carleton/Ottawa	Georgia Fire	D. dipsaci	G-129	Grey		D. dipsaci
B-5	Brant	Music	D. dipsaci	G-136	North Frontenac	Porcelain	D. dipsaci
B-7	Hamilton	Music	D. dipsaci	G-137	Prince Edward	Fish Lake 4 (F4)	D. dipsaci
B-9	Frontenac	Music	D. dipsaci	G-141	Kent	Music	D. dipsaci
B-38	Simcoe	Music	D. dipsaci	G-142	Kent	Music	D. dipsaci
B-39	Simcoe		D. dipsaci	BK-01	Frontenac		D. dipsaci
B-51	Huron	Music	D. dipsaci	JP-01	Windsor-Essex		D. dipsaci
B-52	Huron	Music	D. dipsaci	GE#01	Norfolk		D. dipsaci
B-53	Huron	Music	D. dipsaci	GE#02	Norfolk		D. dipsaci
C-40	Wellington	Music	D. dipsaci	AC#01	Bruce		D. dipsaci
C-58	Perth	Music	D. dipsaci	AC#02	Bruce		D. dipsaci
C-59	Perth	German Red	D. dipsaci	PT-01–02	Middlesex		D. dipsaci
C-60	Wellington	Music	D. dipsaci	SB-01	Bruce		D. dipsaci
C-61	Wellington	Music	D. dipsaci	PM-01	York		D. dipsaci
C-62	Haliburton	Music	D. dipsaci	PL-03a	Niagara		D. dipsaci
C-64	Haliburton	Composite Sample	D. dipsaci	PL-03b	Niagara		D. dipsaci
C-67	Halton	Music	D. dipsaci	PL-03c	Niagara		D. dipsaci
C-68	Halton	Music	D. dipsaci	GL#G	Perth		D. dipsaci
C-69	Halton	Music	D. dipsaci	GL#R	Perth		D. dipsaci
D-70	Perth	Music	D. dipsaci	TH	Rockland	onion	D. dipsaci
D-73	Perth	Anna's	D. dipsaci	B-8			D. destructor

Materials and methods

Nematode populations

One hundred and twenty-three samples, consisting of 10 garlic bulbs each from 79 farms located in 33 counties across Ontario, were collected in 2011. The garlic bulbs were cut into small pieces and nematodes were extracted using the Baermann funnel method (Baermann, 1917). The nematode species’ identity was established using morphological (stylet length, lateral fields, post-uterine sac, etc.) and molecular methods (ITS1 PCR amplified, sequenced and blasted into Genbank). In this study, a total of 90 populations of D. dipsaci isolated from garlic were tested and compared with one from onion and one sample of D. destructor from Ontario (Table 1).

Table 2. Sequences of the RAPD primers used in the study

Code	Sequence
OPG-02	GGCACTGAGG
OPG-03	GAGCCCTCCA
OPG-04	AGCGTGTCTG
OPG-05	CTGAGACGGA
OPG-06	GTGCCTAACC
OPG-11	TGCCCGTCGT
OPG-12	CAGCTCACGA
OPG-13	CTCTCCGCCA
OPG-14	GGATGAGACC
OPG-16	AGCGTCCTCC
OPG-17	ACGACCGACA
OPG-18	GGCTCATGTG
OPG-19	GTCAGGGCAA
RAPD-01	TGGCTGGGCT
RAPD-02	TCAATTCTTC
RAPD-03	TTCTTCTTCC
RAPD-04	CAAGCGTTAC
RAPD-05	TAGATAGTAG
RAPD-06	CGCTTCCGTC
RAPD-07	TGAAGAAACT
RAPD-08	GTGCCGTCAT

DNA extraction

One or two nematodes were handpicked from the extraction and subjected to DNA extraction. The nematodes were crushed in microtubes containing 40 μL 10× PCR buffer (100 mM Tris-HCl, pH 9.0 at 25 °C, 500 mM KCl, 15 mM MgCl₂), 10 μL Proteinase K (1 mg mL⁻¹), 50 μL distilled water. The microtubes were incubated for 1.5 h at 65 °C and 15 min at 95 °C, and stored at −20 °C. DNA concentrations were checked using a NanoDrop spectrophotometer (NanoDrop).

RAPD (random amplified polymorphic DNA)

Twenty-one random primers (Table 2) were screened with the samples TH and G7 which represent D. dipsaci from the onion and garlic populations, respectively, and one primer OPG-05 was selected for the subsequent polymerase chain reaction (PCR). The PCRs were carried out in a 25 μL final volume containing 10 ng of g DNA, 2.5μL 10× PCR buffer, 1.25μL 2.5mM dNTPs, 0.25μL Taq DNA polymerase (Titanium). The amplification programme was: 94 °C for 1 min, 40 cycles of 94 °C for 1 min; 40 °C for 1 min; 72 °C for 2 min, and a final incubation of 72 °C for 10 min. The PCR products were separated by electrophoresis (100V, 1h) in 1.5% agarose gels in TAE buffer with 180–200 ng μL⁻¹ DNA concentration. The RAPD analysis was repeated twice, and gels with clearer bands were used.

Data analysis

The presence or absence of DNA fragments were scored as one and zero, respectively, in the matrix. Simple matching coefficients (SM) (Digby & Kempton, 1987) and hierarchical cluster analysis were performed with NTSYS2.1 using the kimura 2-parameter model. Cluster analysis, by the unweighted pair group method with arithmetic mean (UPGMA), was performed with the SAHN procedure (Exeter Software, Setauket, NY). The robustness of the dendrogram was tested with 1000 bootstrap replications using PAUP software (Swofford, 2003).

Results

Primer screening and RAPD analysis

Of the 21 primers tested with one population of D. dipsaci from onion and one from garlic, primer OPG-05 produced multiple clearly visible bands for both populations and no two bands were of the same size (Fig. 1). The DNA polymorphisms of all 90 populations of D. dipsaci isolated from garlic, one population from onion and one of D. destructor produced with primer OPG-05 are presented in Fig. 2. All populations yielded between 1 and 10 DNA fragments. The size of the fragments ranged from 100–2000 bp with a total of 21 different-sized bands observed. No common fragment was found among all of the populations of D. dipsaci from garlic. Some populations had identical polymorphisms. Only fragments with a high intensity were taken into account.

Fig. 2. RAPD profiles of the 90 Ditylenchus dipsaci populations isolated from garlic, one population from onion and one population of D. destructor.

Fig. 1. RAPD profiles of the 21 primers used for the primer screening (top line: primer ID; second line: nematode samples tested).

Data analysis

The binary matrix and the simple matching coefficient were generated. The average simple matching coefficient among populations of D. dipsaci from garlic was 82%, which was similar to the average similarities when the population from onion and D. destructor were included.

All of the D. dipsaci populations isolated from garlic were grouped into two clusters (Fig. 3): a small cluster consisting of 19 populations from counties in the southern part of the province, including Essex and Kent, and a large cluster with a high degree of genetic similarity (> 0.81) mostly from the eastern region, but also including a few locations from southwestern and northern Ontario. Interestingly, the isolate from onion was the basal to the large cluster. The DNA polymorphisms in D. dipsaci were different from those in D. destructor, which was basal to the two clusters of D. dipsaci. No correlation was found between the DNA polymorphisms and the garlic cultivars.

Fig. 3. Similarity dendrogram of 90 Ditylenchus dipsaci populations constructed from the genetic distance based on random amplified polymorphic DNA fragments, using the un-weighted pair group method (UPGMA).

Discussion

The two distinct clusters of D. dipsaci populations isolated from garlic in Ontario as revealed in this study clearly show significant genetic variation among these populations, which are classified in one biological race of the species. This is the first report of intraspecific genetic variation among populations within a race of D. dipsaci. Previously reported variations using the same method were from populations of the species at the race level (Esquibet et al., 1998, 2003). Based on the ribosomal DNA ITS sequences generated for the species identification used in this study, no significant variations were found from those selected populations (results not shown), which was in agreement with the results from similar studies (Subbotin et al., 2003, 2005).

The intra-racial genetic variation, although not high with an average similarity of coefficient at 82%, is somewhat surprising considering that the populations were from one province, some samples came from neighbouring farms and garlic cloves used as seed has been exchanged across the province. The only other species of nematode where intra-racial variations have been reported are the potato cyst nematodes G. rostochiensis and G. pallida (Sedlak et al., 2004; Blok et al., 1997, 1998), but the populations tested in those studies came from different countries on different continents. It is interesting to note that before the species of G. pallida was established in 1973 (Stone, 1973), it was considered to be a pathotype of G. rostochiensis for several decades (Wollenweber, 1923; Behrens, 1975). This is similar to the situation with D. gigas, which was considered a race of D. dipsaci until 2011 (Vovlas et al., 2011). Some researchers have speculated that both G. pallida and D. gigas are newly evolved species (Dr. S. Subbotin, personal communication). The stem and bulb nematode, like the potato cyst nematode, is a very serious pest, and therefore has been subjected to enormous pressure from human activity e.g. monoculture of resistant cultivars, which is likely a cause for the variation reported here. Intra-racial genetic variations have been widely observed in plant pathogens. These variations may have also contributed to the taxonomic controversies surrounding the species. The grouping of these populations in two clusters seems to suggest that there were two different sources of infestation. Because of the finding that the isolate from onion was basal to the large cluster only, and this cluster consisted of all isolates from the eastern region where D. dipsaci in onion has been reported (Fushtey & Kelly, 1975), it is possible that these populations originated from the onion isolate. Is it possible that those isolates from the southern counties had a different origin? More studies are needed to answer the question.

Race determination for D. dipsaci is essential for successful management of this pest. At the present time, there are no quick and reliable molecular tools to differentiate the races. The genetic variability within a race, as revealed in this study, indicates that such tools would be hard to develop. Several projects are underway in Canada to gain a further understanding of the genetics of D. dipsaci and implement management options, including: (1) sequencing of the complete genomes and transcriptomes of D. dipsaci and D. destructor; (2) phenotyping the populations of D. dipsaci from garlic in Ontario to determine if associations are found with the pattern of genetic variations, and (3) the development and implementation of a clean seed programme.

Acknowledgements

The authors wish to thank those participating garlic farmers for sending the garlic samples for this study.