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Citation Information : Journal of Nematology. Volume 51, Pages 1-14, DOI: https://doi.org/10.21307/jofnem-2019-074
License : (CC-BY-4.0)
Received Date : 01-April-2019 / Published Online: 04-December-2019
The diversity and seasonal fluctuations of plant-parasitic nematodes (tylenchids) were investigated in five alfalfa fields in five counties of the Kerman Province of Iran during four consecutive seasons in 2013. Hundred soil samples were obtained per county, with 25 sub-samples per field being composited to five to represent five replicates per field per county per season. In total, 500 samples were analyzed during the study. Nematodes were extracted from soil samples using the modified tray method. In total, 11 plant-parasitic nematode genera and 12 species were recorded. According to prominence values,
Worldwide, alfalfa (Medicago sativa L.), a flowering plant belonging to the family Fabaceae, is cultivated as a forage crop (Tucak et al., 2008). It is the most important forage crop in Iran due to its superior feeding value for cattle, which is the main meat/protein food source (Tucak et al., 2008). The genus Medicago comprises many species, with up to 23 being cultivated in Iran (Ghanavati et al., 2007).
A wide range of plant-parasitic nematodes have been associated with alfalfa crops in various countries, such as the USA (Gray and Griffin, 1994), South Africa (Kleynhans et al., 1996) and others (Abivardi and Sharafeh, 1973; Sturhan and Brzeski, 1991). Moreover, nematodes such as Aphelenchoides ritzemabosi and Ditylenchus dipsaci are major pests of the foliar parts of alfalfa (Gray et al., 1994; Milano de Tomasel and McIntyre, 2001), whereas Meloidogyne spp. and Pratylenchus spp. in particular infect roots of this genus and cause substantial yield losses (Hafez and Sundararaj, 2009). In Idaho (USA), D. dipsaci infections inflicted reduction in total yield of alfalfa ranging from 6 to 13% (Hafez, 1998). This author also reported yield reductions of between approximately 0.3 and 6% in the same study as a result of parasitism by M. hapla. Ditylenchus dipsaci is considered the most damaging plant-parasitic nematode that parasitize alfalfa in Iran (Kheiri, 1972; Abivardi and Sharafeh, 1973). Ditylenchus dipsaci was the main cause of yield loss in alfalfa fields in the Khafr County, with stunted and infected plants being visible as patches of poor growth within the field (Kheiri, 1972; Abivardi and Sharafeh, 1973). Worldwide, nematode pests associated with roots of alfalfa include Meloidogyne spp., Xiphinema spp., Pratylenchus spp. and Helicotylenchus spp. (Hassanzadeh et al., 2004, Westerdahl and Frate, 2007; McCord, 2012). These nematode genera are also dominant in alfalfa fields in Iran.
Soil nematode communities represent superior biological tools for evaluating soil quality and plant health in terrestrial ecosystems (Wang et al., 2009; Pen-Mouratov et al., 2010). Nematode biodiversity in different soil habitats had been studied widely (Potter and McKeown, 2003; Biederman and Boutton, 2009; Zhang et al., 2012) as a crucial research component that gives an indication of soil quality (Bongers, 1990; Yeates, 2003; Neher et al., 2005). The latter is important for sustainable agriculture and also constitutes one of the main aims of an ecological study. Alfalfa is the main food source of domestic animals in the Kerman Province of Iran, which is in return the main income source of producers. Understanding distribution of most dominant plant-parasitic nematodes on alfalfa in Iran would provide better crop protection recommendations to alfalfa growers in Kerman Province, the main alfalfa production area, of Iran.
Hence, the aim of this investigation was to determine the biodiversity, prominence and seasonal population fluctuations of plant-parasitic tylenchids that occur in soil of alfalfa plants in this province and to identify whether relationships exist between selected soil properties and nematode population densities.
Five alfalfa fields in each of five counties, namely, Bam, Jiroft, Bardsir, Rabor and Rigan, were sampled for the presence of plant-parasitic nematodes during 2013 and 2014. Selection of the counties was based on the alfalfa production area of the province (Fig. 1; Table 1). Soil samples were collected four times during the year, namely, in October (Autumn), March (Winter), June (Spring) and August (Summer). In each field in each county, five discrete sub-samples were collected from each of five independent, 10 m × 10 m plots, representing five replicates and randomly chosen per field. The five sub-samples taken per field per plot were added together, mixed and one homogenized composite sample (representing one replicate) per plot ultimately obtained, totaling five replicates per field. In total, 500 samples (five composite samples per field × five fields × four seasons) were analyzed for nematode counts and identification. After removing the aboveground plant debris, soil samples were collected from the soil of alfalfa plants using a soil core with a 5-cm-diameter opening (Zhang et al., 2012).
Soil samples were stored in individual plastic bags, kept at 4°C and processed within one week after sampling. Additional soil was obtained during nematode sampling and used to analyze soil pH and electrical conductivity (EC) (Zhang et al., 2012) using standard methods (Rowell, 1994) (Table 1). Also, soil structure (% clay, % sand and % silt) was determined (Bouyoucos, 1962; Beretta et al., 2014). Average means for rainfall and temperature (Hashemi Nasab Khabisi et al., 2013; Kavian et al., 2016) as listed in Table 1, were used. The climate in the province ranges from dry and cold (Rabor and Bardsir counties) to warm and humid (Bam, Jiroft and Rigan counties) (Jalali-Far et al., 2012).
Nematodes were extracted from 100 cm3 composite soil samples over 72 hr using 40 × 25-cm plastic trays according to the modified Baermann tray technique (Whitehead and Hemming, 1965; Spaull and Braithwaite, 1979). The nematodes were counted with a stereomicroscope (Olympus CH-2; Japan) and their genera identification finalized using a light microscope (Nikon Eclipse E200). Nematodes were then fixed with a hot 4% formaldehyde solution and transferred to anhydrous glycerin (De Grisse, 1969) for species identification. The nematode genera were identified according to the classification (Brzeski, 1991; Andrássy, 2005; Castillo and Vovlas, 2007; Geraert, 2008, 2011). In addition, for accurate diagnosis of Meloidogyne, Pratylenchus, Merlinius and Ditylenchus species, DNA extraction was done using the Chelex method (Straube and Juen, 2013). The nematodes were identified using 28 S rDNA marker according to the protocol provided by Shokoohi et al. (2018).
The relationships between nematode population density (MPD) and frequency of occurrence (FO) of each nematode genus identified were expressed as prominence value (PV) for each county and season. Ultimately, to determine which genera were predominant in the Kerman Province in alfalfa bulk soil, population density data were pooled for each genus across the counties and over the four seasons and PV, MPD and FO again calculated using the equation PV = Population density × FO/10 (Bolton et al., 1989; De Waele and McDonald, 2000).
In addition, mean population densities for each nematode genus were log-transformed (log10(x + 1)) and subjected to repeated Measures Analyses of Variance using SPSS Version 24 (IBM, 2016) to determine whether localities and/or seasons influenced abundance. Nematode biodiversity indices, representing the Evenness Index (E) (Zeng et al., 2012) and Shannon Index (H’) (Colwell, 2009) were calculated. The E index refers to homogeneity of the species, whereas the H’ index is the most popular biodiversity index used to summarize the diversity of a population to which each member belongs according to a unique group. The latter index also takes into account species richness and the proportion of each species within the community studied. Finally, correlations between nematode abundance and the two selected soil parameters, pH and EC were analyzed through two-tailed Pearson correlation using SPSS 24 (IBM 2016). Correlation of the localities with rainfall, temperature, pH and EC of the soil was done using XLSTAT (Addinsoft, 2007) through principal component analysis (PCA).
In total, 11 plant-parasitic nematode genera (Table 1 or Fig. 2A-K) and 12 species were identified from 25 fields sampled from five counties. These included Ditylenchus acutus, D. terricolus, D. myceliophagus (Shokoohi et al., 2018), D. savarae (Shokoohi et al., 2018), Helicotylenchus pseudorobustus, Meloidogyne javanica, Merlinius brevidens, Nanidorus minor, Pratylenchus cruciferus, P. neglectus, Amplimerlinius globigerus and Scutylenchus rugosus. Unidentified species, Aphelenchoides, Paratylenchus and Rotylenchus, were also present.
The results indicated that locality and season effects significantly (p ≤ 0.01) affected nematode community (Table 2). In terms of locality and seasonal fluctuation of nematode genera, substantial variation existed for MPD in different nematode genera over localities and four seasons (Table 3; Fig. 2A-K). According to PV, alfalfa fields in Bardsir were dominated by H. pseudorobustsu during the Winter (Table 3; Fig. 2B) and by P. neglectus during Summer (Table 3; Fig. 2C), whereas M. brevidens dominated in fields at Rabor during winter (Table 3; Fig. 2E). Ditylenchus dominated in alfalfa fields in Jiroft during Autum and Winter (Table 3; Fig. 2A) and Meloidogyne during Spring (Table 3; Fig. 2D). Interestingly, some genera were not detected at some localities and/or during some seasons. For example, P. neglectus was not present at Jiroft (Table 3; Fig. 2C), and neither Meloidogyne at Bardsir and Rigan (Table 3; Fig. 2D), A. globigerus at Rabor and Rigan (Table 3; Fig. 2F), P. neglectus at Jiroft (Table 3; Fig. 2H), S. rugosus at Bardsir, Rabor and Jiroft (Table 3; Fig. 2I), Nanidorus minor at Rabor and Jiroft (Table 3; Fig. 2J), and Rotylenchus sp. at Rabor, Bardsir, Jiroft and Rigan (Table 3; Fig. 2K). For seasons, the same phenomenon applied to M. javanica that were not detected during Autumn (Table 3; Fig. 2D), N. minor during Winter (Table 3; Fig. 2J), Rotylenchus sp. during Summer (Table 3; Fig. 2K) and S. rugosus during Autumn (Table 3; Fig. 2I) in all counties.
When nematode data were pooled for the five counties and four seasons, the predominant plant-parasitic nematode genera identified from the soil of alfalfa in the Kerman Province according to PV were Ditylenchus spp. (e.g. D. acutus, D. myceliophagus, D. terricolus and D. sarvarae), followed by H. pseudorobustus, P. neglectus and M. javanica, whereas the least dominant was Rotylenchus sp. (Table 4). Ditylenchus spp. had the highest occurrence, whereas Rotylenchus sp. and S. rugosus occurred least among the samples.
Nematode biodiversity indices (H’ or E) differed significantly (p ≤ 0.05) among counties (Table 5), where H’ and E values were highest in Bam and lowest in Jiroft. A significant, negative correlation existed for soil pH and the abundance of S. rugosus (r = −0.964; p ≤ 0.01), whereas significant and positive correlations existed between soil EC and the abundance of H. pseudorobustus (r = 0.89; p ≤ 0.05), Aphelenchoides sp. (r = 0.92; p ≤ 0.05), A. globigerus (r = 0.97; p ≤ 0.01) and P. neglectus (r = 0.90; p ≤ 0.05) (Table 6). The PCA was performed to study the correlation of the temperature and rainfall with the counties (Fig. 3). An accumulated variability of 94.62% was detected in the analysis which was 59.53% for F1 and 35.09% for F2. The active variables including rainfall (–0.83) and EC (–0.85) had negative correlation to F1, whereas temperature (0.98) and pH (0.12) had positive correlation to F1. Rainfall (–0.44) and temperature (–0.05) had negative correlation to F2, whereas EC (0.52) and pH (0.97) had positive correlation to F2. The results suggested that in Bardsir county EC affected diversity of plant-parasitic nematodes more than in other counties, whereas in Rigan county pH was the most effective factor on nematode diversity. In the counties Jiroft and Bam, temperature played an important role, whereas in Rabor county rainfall played an important role in the diversity of plant-parasitic nematodes (Fig. 3).
The study provided baseline information on plant-parasitic nematode diversity associated with alfalfa bulk soil in the south eastern, Kerman Province of Iran. Among the identified 11 plant-parasitic nematode genera and 12 species, the predominant genera in descending order were Ditylenchus, Helicotylenchus, Pratylenchus and Meloidogyne, which have also been recorded in alfalfa cultivated in the East Azarbaijan Province of Iran (Alavi and Barooti, 1995; Eskandari et al., 2015) and other countries (Goodell and Ferris, 1980; Marais, 1990; Westerdahl and Frate, 2007; McCord, 2012). Interestingly, the predominant genus in the soil, Ditylenchus, with identification of a new species D. savarae (Shokoohi et al., 2018), was associated with alfalfa in the Kerman Province. Helicotylenchus pseudorobustus, second in predominance, was reported as the most abundant in alfalfa fields in Colorado, USA (Simmons et al., 2008), in Oman, South Africa and other countries (Marais, 1990; Mani and AL Hinai, 1996). Pratylenchus neglectus, as the third predominant genus identified in the current study, was the most commonly occurring genus associated with alfalfa in Oman (Mani and Al Hinai, 1996) and has also been reported in South Africa and other countries (Marais, 1990; Simmons et al., 2008). Meloidogyne javanica, generally low in abundance in the soil of alfalfa in our study except for Jiroft, being fourth in predominance, was also reported as abundant in alfalfa fields in Oman (Mani and Al Hinai, 1996) and South Africa (Marais, 1990). Merlinius brevidens, fifth in predominance, also occurred in the soil samples from all counties sampled in the Kerman Province and has been reported from alfalfa fields in other countries (Westerdahl and Frate, 2007).
The production area under investigation during our study covered 180 726 km2 with different abiotic factors such as climate (e.g. temperature and rainfall) (Ferris et al., 2012; Hashemi Nasab Khabisi et al., 2013; Kavian et al., 2016), elevation (Kergunteuil et al., 2016) and edaphic variables (Sarreshtehdari, 2002), which could impact the nematode assemblages. Such factors are major determinants of survival and reproduction of plant-parasitic nematodes since they affect nematode occurrence, population densities and the degree of symptom development and expression in infected hosts (Amarasena et al., 2016). Seasonal fluctuations of nematode population densities are common in alfalfa ecosystems, varying among localities and over years (Norton, 1963; Williams-Woodward and Gray, 1999; Simmons et al., 2008). The plant-parasitic nematode diversity associated with alfalfa in the Iranian counties over four consecutive seasons suggested that the environmental conditions played an important role in nematode ecology. This phenomenon was most pronounced for the abundance of Merlinius, showing a significant interaction for county × season for Merlinius and Pratylenchus among the five counties, and for Merlinius and Ditylenchus for seasons. Although no correlation statistics was done for climatic variables during our study due to such data not being recorded at each field for each county, the seasonal effect was most probably one of the major contributors to the predominance of the genera at localities. This was premised on the basis that climatic conditions in the province ranged from dry and cold (Rabor and Bardsir counties) to warm and humid (Bam, Jiroft and Rigan counties) (Jalali-Far et al., 2012). For example, data from our study showed that during winter Helicotylenchus pseudorobustus and Merlinius brevidens dominated at Bardsir and Rabor when minimum temperatures lower than 5°C prevailed (Hashemi Nasab Khabisi et al., 2013; Kavian et al., 2016). Nonetheless, for Helicotylenchus these findings contradicted reports stating that population densities of the genus decreased at 5 to 10°C, with optimum temperature for development, reproduction and survival being from 20 to 30oC (Azmi, 1979; Nath et al., 1998). Similarly, Merlinius also prefers moderate temperatures of 20oC (Malek, 1980), which is in contrast to the predominance of this genus in winter at Bardsir and Rabor, where temperatures lower than 5°C prevailed. Pratylenchus neglectus on the other hand was dominant in summer at Bardsir at a higher mean temperature within 25 and 30oC being preferred by species of this genus to support their optimal biological functions (Griffin and Gray, 1990; Mizukubo and Adachi, 1997). Dominance of Ditylenchus species in Jiroft during Autum and Winter and M. javanica during Spring again accentuated the preference of species of these genera to optimally develop and reproduce at relatively mild temperature ranges and in areas with relatively high rainfall (Loubser and Meyer, 1987; Morris et al., 2011; Hajihassani, 2016; Hajihassani et al., 2017). The absence of genera at some localities and during some seasons, e.g. Meloidogyne species not occurring at Bardsir (low minimum and mild to high maximum temperatures) and Rigan (mild to high temperature range), and not during Autumn at all five localities, is another phenomenon emanating from our study for which an explanation could not be given at this stage, and warrants further investigation. The PCA also showed the diversity of nematode in Rabor as being correlated with rainfall, which is in accordance with the rainfall value of the county. This result is in agreement with Munteanu (2017), which indicated that rainfall and temperature positively affected the diversity of nematodes in Norway.
Concerning soil texture, significant, positive correlations recorded between the abundance of H. digonicus and M. brevidens in alfalfa fields in California (USA) and fine-textured soils, while that of Meloidogyne arenaria showed negative but significant correlations with this soil type (Goodell and Ferris, 1980). However, no deductions can be made on this topic from our study, but it is interesting that H. pseudorobustus and Merlinius brevidens were dominant in sandy-loam soils in Bardsir and Rabor, and Meloidogyne javanica in sandy-loam soils in Jiroft.
The biodiversity indices H’ and E, which are popular and useful tools for studying different factors and their effects on nematode populations, revealed seasonal effects regarding the diversity of the plant-parasitic nematode genera identified as a result of our study. A higher H’ and E value, for nematode assemblages from alfalfa fields in the Bam, and a slightly lower value for the Jiroft accentuated the effects that different abiotic and/or biotic factors may have on nematode assemblages. These indices, however, varied among the counties suggesting that a greater diversity and more even distribution of the prevalent nematode genera and species occurred at Bam, Bardsir, Rabor and Rigan compared to Jiroft. This scenario especially applies for the H’ index, which is suggested to be used in Iran.
High levels of the selected soil chemical properties determined in our study, namely, pH and EC, have been reported to result in decreased population densities of plant-parasitic nematode populations. No correlation was apparent for soil pH and nematode genera identified during our study, except for Scutylenchus with a high negative correlation with pH, since values generally ranged in the neutral zone (7.51–7.59) (McCauley et al., 2017) for the other genera. Matute (2013), however, reported that pH levels between 5 and 6 negatively correlated with plant-parasitic nematodes abundance in the rhizosphere of Brassica rapa in central Arkansas (USA). In addition, Burns (1971) reported significantly higher abundance of Tylenchinae and Hoplolaimus galeatus, associated with soybean in the USA, at pH 6 than at pH 4 or 8. Concerning EC, significant correlations for Helicotylenchus and Pratylenchus and this variable is in agreement with results by Mendoza et al. (2008). These authors showed a positive correlation between soil EC and low nematode abundance in Nebraska (USA). It is important to note that other soil chemical properties such as calcium, iron, organic matter and nitrogen (Mateille et al., 2014) could have impacted on nematode population densities but were not determined during our study and should be included in future research on this topic.
County Bam, with a higher quality of alfalfa (Tadayyon and Zafarian, 2016), also had the highest plant-parasitic nematode diversity, and therefore, crop production practices known to promote high plant-parasitic diversity should be practiced. Environmental conditions were shown to affect the diversity of plant-parasitic nematodes in Kerman Province, as shown by the PCA, but detailed studies on this topic are needed. Breeding of alfalfa cultivars with resistance to economically important nematode pests such as Meloidogyne and Pratylenchus species should be considered to protect alfalfa crops in Kerman Province. Future studies should also establish whether economically important nematodes of aerial parts on alfalfa are present in the province.