Diversity and seasonal fluctuation of tylenchid plant-parasitic nematodes in association with alfalfa in the Kerman Province (Iran)

Nematode extraction and identification

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).

Statistical analyzes

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).

Results

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.

Figure 2:

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.

Discussion

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 30^oC (Azmi, 1979; Nath et al., 1998). Similarly, Merlinius also prefers moderate temperatures of 20^oC (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 30^oC 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.

Conclusion

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.