Worldwide, alfalfa (
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
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).
List of the localities sampled in the Kerman Province of Iran for the presence of nematodes, physical and chemical characteristics of the soils as well as rainfall and temperature data.
Chemical soil characteristics | Temperature (°C) for the sampling period | ||||||
---|---|---|---|---|---|---|---|
County | GPS coordinates | pH | EC (mhos/cm) | Soil type | Mean rainfall (mm) for the study period | Minimum | Maximum |
Bam | N29°06′22″; E58°21′25″ | 7.53 ± 0.06 | 1.74 ± 0.2 | Sandy | 74 | 17 | 29 |
Bardsir | N29°55′39″; E56°34′20″ | 7.59 ± 0.08 | 2.97 ± 0.7 | Sandy-loam | 148 | 3 | 26 |
Jiroft | N28°40′41″; E 57°44′26″ | 7.53 ± 0.02 | 1.64 ± 0.06 | Sandy-loam | 155 | 17 | 33 |
Rabor | N29°17′28″; E56°54′47″ | 7.51 ± 0.03 | 2.04 ± 0.08 | Sandy-loam | 243 | 1 | 24 |
Rigan | N28°38′58″; E59°1′15″ | 7.59 ± 0.05 | 1.86 ± 0.04 | Sandy | 55 | 19 | 34 |
EC and pH data obtained from 20 samples per field ±SE. Mean of rainfall and temperature obtained from Hashemi Nasab Khabisi et al. (2013) and Kavian et al. (2016).
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
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
The results indicated that locality and season effects significantly (
MANOVA of effect of the locality and season on nematode community using SPSS 24.
Source | Type III sum of squares | df | Mean square |
|
Sig. |
---|---|---|---|---|---|
Corrected model | 596,437.917a | 19 | 31,391.469 | 423.827 | 0.000 |
Intercept | 3827,900.417 | 1 | 3827,900.417 | 51,681.824 | 0.000 |
Location | 45,860.167 | 4 | 11,465.042 | 154.794 | 0.000 |
Season | 166,211.917 | 3 | 55,403.972 | 748.028 | 0.000 |
Location × Season | 384,365.833 | 12 | 32,030.486 | 432.455 | 0.000 |
Error | 2,962.667 | 40 | 74.067 | ||
Total | 4427,301.000 | 60 | |||
Corrected Total | 599,400.583 | 59 |
a
Prominence values (PV), mean population density (MPD) and frequency of occurrence (FO) (%) of plant-parasitic nematode species that were identified in association with alfalfa in five counties in the Kerman Province of Iran during four consecutive seasons (Fall, Winter, Spring and Summer) of 2013.
Bam | Rabor | Rigan | Bardsir | Jiroft | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Species | PV | MPD | FO (%) | PV | MPD | FO (%) | PV | MPD | FO (%) | PV | MPD | FO (%) | PV | MPD | FO (%) |
|
|||||||||||||||
Season 1 | 43 | 48 | 80 | 30 | 30 | 100 | 60 | 60 | 100 | 19 | 21 | 83 | 366 | 366 | 100 |
Season 2 | 94 | 94 | 100 | 103 | 103 | 100 | 120 | 120 | 100 | 59 | 65 | 83 | 344 | 344 | 100 |
Season 3 | 83 | 93 | 80 | 53 | 53 | 100 | 137 | 137 | 100 | 95 | 95 | 100 | 107 | 107 | 100 |
Season 4 | 71 | 79 | 80 | 72 | 72 | 100 | 36 | 36 | 100 | 76 | 76 | 100 | 8 | 11 | 50 |
|
|||||||||||||||
Season 1 | 29 | 32 | 80 | 16 | 18 | 75 | 29 | 32 | 80 | 64 | 64 | 100 | 2 | 3 | 25 |
Season 2 | 40 | 64 | 40 | 29 | 29 | 100 | 21 | 27 | 60 | 81 | 81 | 100 | 2 | 3 | 25 |
Season 3 | 2 | 4 | 20 | 31 | 31 | 100 | 60 | 78 | 60 | 59 | 84 | 50 | 3 | 4 | 50 |
Season 4 | 7 | 11 | 40 | 33 | 38 | 75 | 1 | 3 | 20 | 3 | 5 | 33 | 3 | 4 | 50 |
|
|||||||||||||||
Season 1 | 1 | 3 | 20 | 4 | 5 | 75 | 23 | 36 | 40 | 18 | 25 | 50 | 0 | 0 | 0 |
Season 2 | 5 | 8 | 40 | 31 | 36 | 75 | 4 | 7 | 40 | 17 | 24 | 50 | 0 | 0 | 0 |
Season 3 | 0 | 0 | 0 | 4 | 6 | 50 | 2 | 4 | 20 | 4 | 6 | 50 | 0 | 0 | 0 |
Season 4 | 0 | 0 | 0 | 78 | 90 | 75 | 0 | 0 | 0 | 109 | 120 | 83 | 0 | 0 | 0 |
|
|||||||||||||||
Season 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 2 | 25 | 40 | 40 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 3 | 13 | 30 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 34 | 68 | 25 |
Season 4 | 13 | 30 | 20 | 3 | 6 | 25 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
|
|||||||||||||||
Season 1 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 5 | 40 | 10 | 25 | 16 | 4 | 7 | 25 |
Season 2 | 15 | 35 | 20 | 120 | 120 | 100 | 3 | 4 | 40 | 10 | 25 | 16 | 4 | 7 | 25 |
Season 3 | 0 | 0 | 0 | 4 | 7 | 25 | 1 | 3 | 20 | 10 | 25 | 16 | 2 | 4 | 25 |
Season 4 | 2 | 5 | 20 | 2 | 4 | 25 | 0 | 0 | 0 | 10 | 25 | 16 | 0 | 0 | 0 |
|
|||||||||||||||
Season 1 | 5 | 6 | 60 | 0 | 0 | 0 | 0 | 0 | 0 | 0.5 | 1 | 16 | 2 | 3 | 25 |
Season 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 6 | 33 | 2 | 3 | 25 |
Season 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 40 | 69 | 33 | 0 | 0 | 0 |
Season 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 11 | 20 | 33 | 0 | 0 | 0 |
|
|||||||||||||||
Season 1 | 1 | 1 | 80 | 7 | 7 | 100 | 3 | 4 | 60 | 1 | 1 | 83 | 1 | 2 | 50 |
Season 2 | 11 | 12 | 80 | 4 | 4 | 100 | 1 | 2 | 60 | 1 | 1 | 83 | 1 | 2 | 50 |
Season 3 | 4 | 4 | 80 | 7 | 7 | 100 | 15 | 15 | 100 | 12 | 14 | 83 | 2 | 3 | 75 |
Season 4 | 4 | 4 | 80 | 5 | 5 | 100 | 7 | 8 | 80 | 5 | 7 | 50 | 13 | 13 | 100 |
|
|||||||||||||||
Season 1 | 0.5 | 1 | 20 | 4 | 5 | 75 | 13 | 17 | 60 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 2 | 0 | 0 | 0 | 28 | 40 | 50 | 3 | 5 | 40 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 3 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 3 | 20 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 2 | 16 | 0 | 0 | 0 |
|
|||||||||||||||
Season 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 2 | 6 | 14 | 20 | 0 | 0 | 0 | 4 | 6 | 40 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 3 | 21 | 27 | 60 | 0 | 0 | 0 | 3 | 6 | 20 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 4 | 3 | 7 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
|
|||||||||||||||
Season 1 | 5 | 6 | 60 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 3 | 1 | 2 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 4 | 0 | 0 | 0 | 0 | 0 | 0 | 10 | 23 | 20 | 0.5 | 1 | 16 | 0 | 0 | 0 |
|
|||||||||||||||
Season 1 | 0.5 | 1 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 2 | 2 | 3 | 40 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 3 | 1 | 2 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Season 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
aincluding
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
Prominence values (PV), mean population density (MPD) and frequency of occurrence (FO) (%) of plant-parasitic nematode species identified in association with alfalfa pooled for five counties in the Kerman Province of Iran and the four consecutive seasons (Fall, Winter, Spring and Summer) of 2013.
Nematode species | Prominence value (PV) | Mean population density (MPD) | Frequency of occurrence (FO: %) |
---|---|---|---|
|
89 | 93 | 92 |
|
18 | 24 | 59 |
|
18 | 30 | 34 |
|
10 | 41 | 6 |
|
8 | 18 | 21 |
|
6 | 18 | 12 |
|
4 | 6 | 41 |
|
3 | 8 | 16 |
|
3 | 12 | 7 |
|
2 | 6 | 11 |
|
1 | 3 | 7 |
Nematode biodiversity indices (H’ or E) differed significantly (
Nematode community indices for the five counties sampled during four consecutive seasons in the Kerman Province of Iran during 2013.
County | Shannon index (H’) | Evenness index |
---|---|---|
Bam | 2.4 ± 0.02 | 0.83 ± 0.02 |
Bardsir | 2.2 ± 0.03 | 0.73 ± 0.03 |
Jiroft | 1.0 ± 0.01 | 0.58 ± 0.01 |
Rabor | 2.1 ± 0.02 | 0.74 ± 0.02 |
Rigan | 2.1 ± 0.02 | 0.71 ± 0.02 |
Ranges are mean±standard error.
Correlation data for pH and electrical conductivity (EC) and plant-parasitic nematode speciesa identified in association with alfalfa from five counties in the Kerman Province of Iran during 2013.
Helicoty-lenchus | Dityle-nchus | Amplim-erlinius | Pratyle-nchus | Aphelen-choides | Rotyle-nchus | Nani-dorus | Paratyl-enchus | Scutyle-nchus | Merli-nius | Meloid-ogyne | pH | EC | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Helicotylenchus | 1 | −0.334 | 0.838 | 0.764 | 0.941* | 0.111 | −0.156 | −0.101 | −0.045 | −0.114 | −0.528 | 0.1 | 0.885* |
Ditylenchus | −0.334 | 1 | 0.122 | −0.318 | −0.113 | −0.689 | 0.212 | −0.638 | −0.341 | −0.58 | 0.259 | 0.282 | −0.06 |
Amplimerlinius | 0.838 | 0.122 | 1 | 0.783 | 0.872 | 0 | −0.388 | −0.355 | −0.295 | −0.15 | −0.361 | 0.236 | 0.972** |
Pratylenchus | 0.764 | −0.318 | 0.783 | 1 | 0.799 | 0.276 | −0.584 | 0.294 | −0.501 | 0.426 | −0.674 | 0.44 | 0.902* |
Aphelenchoides | 0.941* | −0.113 | 0.872 | 0.799 | 1 | −0.159 | −0.067 | −0.069 | −0.349 | −0.171 | −0.7 | 0.411 | 0.917* |
Rotylenchus | 0.111 | −0.689 | 0 | 0.276 | −0.159 | 1 | −0.747 | 0.274 | 0.433 | 0.703 | 0.287 | −0.571 | 0.083 |
Nanidorus | −0.156 | 0.212 | −0.388 | −0.584 | −0.067 | −0.747 | 1 | −0.143 | 0.139 | −0.706 | −0.104 | 0.097 | −0.435 |
Paratylenchus | −0.101 | −0.638 | −0.355 | 0.294 | −0.069 | 0.274 | −0.143 | 1 | −0.359 | 0.781 | −0.568 | 0.398 | −0.128 |
Scutylenchus | −0.045 | −0.341 | −0.295 | −0.501 | −0.349 | 0.433 | 0.139 | −0.359 | 1 | −0.253 | 0.688 | −0.964** | −0.368 |
Merlinius | −0.114 | −0.58 | −0.15 | 0.426 | −0.171 | 0.703 | −0.706 | 0.781 | −0.235 | 1 | −0.194 | 0.108 | 0.027 |
Meloidogyne | −0.528 | 0.259 | −0.361 | −0.674 | −0.7 | 0.287 | −0.104 | −0.568 | 0.688 | −0.194 | 1 | −0.802 | −0.522 |
pH | 0.1 | 0.282 | 0.236 | 0.44 | 0.411 | −0.571 | 0.097 | 0.398 | −0.964** | 0.108 | −0.802 | 1 | 0.322 |
EC (mhos/cm) | 0.885* | −0.06 | 0.972** | 0.902* | 0.917* | 0.083 | −0.435 | −0.128 | −0.368 | 0.027 | −0.522 | 0.322 | 1 |
aThe species including
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
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
Concerning soil texture, significant, positive correlations recorded between the abundance of
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
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