Identification of HG Types of Soybean Cyst Nematode Heterodera glycines and Resistance Screening on Soybean Genotypes in Northeast China

Soil sampling was conducted at soybean harvest time in two regions, Anda (N:46^o24′, E:125^o22′) and Wuchang (N:44^o44′, E:127^o14′), of Heilongjiang Province where SCN was present and increased virulence on resistant cultivars was known. Each soil sample contained more than 20 cores taken down to 20 cm depth with a 2.5-cm-diameter soil probe. The mixed soil samples were kept at 4^oC and five cysts were isolated from each soil sample. Then each cyst was cultured on susceptible ‘Dongsheng1’ as the first generation (Hu et al., 2017) in 12-cm-diameter × 8-cm-deep plastic pots containing a 1:1 ratio of sand to soil under controlled greenhouse condition at 23°C to 28°C with 16-hr photoperiod. After 35 to 40 days, one single cyst was collected from the roots and the second generation was initiated. If there was no reproduction, the pot was discarded. To obtain a near-homogeneous population for producing more stable reactions on soybean plants, this process was continued for more than five generations.

Indicator lines PI 548402 (‘Peking’, indicator line #1), PI 88788 (#2), PI 90763 (#3), PI 437654 (#4), PI 209332 (#5), PI 89772 (#6), PI 548316 (‘Cloud’, #7) were utilized for HG Type determination (Niblack et al., 2002). ‘Pickett’ was added to identify the different SCN virulence profiles. Eighteen soybean cultivars/germplasms (Table 1) were utilized for resistance evaluation against SCN populations, including one wild type G. soja ‘ZYD00006’, three breeding lines, 11 cultivars developed primarily for resistance to HG Type 0- by Heilongjiang Academy of Agricultural Sciences in northeast China (Li et al., 1998; Liu, 2005; Tian et al., 2007; Wu et al., 2011; Song, 2012; Wang, 2013; Yu et al., 2013), and three main local commercial cultivars (‘Hefeng25’, ‘Suinong14’, ‘Dongsheng1’).

Nematode inoculum was prepared from soybean plants infected for more than 35 days in the greenhouse. Cysts of H. glycines from roots and soil were extracted by hand over nested sieves. First, soil with roots was poured into 1-liter beaker containing water. After the mixture was vigorously stirred with a glass rod, it was decanted through an 800 µm pore sieve nested on a 150 µm pore sieve. After rinsing under a strong jet of water, the debris and materials on the 150 µm pore sieve were poured onto Whatman No. 4 filter paper which was supported by a 12.5-cm-diameter funnel. The filter paper was dried at room temperature. Most cysts were around the outer layer of the filter paper. The cysts were collected and were crushed over a 75 µm pore sieve nesting on a 25 µm pore sieve with hand-decanting to release eggs (Schmitt and Shannon, 1992). Eggs collected on the 25 µm sieve were incubated at 28°C for three or five days to obtain second-stage juveniles (J₂) as inoculum.

To determine SCN HG Types and to evaluate resistant sources, plants were grown in 12-cm-diameter × 8-cm-deep plastic pots containing a 1:1 ratio of sand to soil under controlled greenhouse condition at 23 ^oC to 28^oC with 16 hr of photoperiod. Two seeds were planted in each pot and thinned to one seedling after germination. Each treatment was replicated with five individual plants. Two holes around each seven-day-old plant were made with a 1 ml pipette tip and 0.5 ml water suspension containing 400 J₂ was injected into each hole (total 800 J₂ per plant). Then the holes were covered with soil. Plants were arranged on benches according to completely randomized design in the greenhouse. Twenty-one days after inoculation, white females were dislodged by washing plant roots individually under a strong jet of water over 800 µm and 150 µm aperture sieves. After females/cysts per plant were counted, eggs per plant were released over 75 µm and 25 µm aperture sieves as described above. Female index (FI) was calculated as follows: FI = (average number of females found on indicator lines or soybean genotypes/average number of females found on susceptible line Lee 68) × 100. Each experiment was repeated two times (Test 1 and Test 2).

For HG Type identification, an FI equal to or greater than 10 on any indicator line or soybean genotype was assigned as susceptible, and as resistant when FI < 10. HG Type determination was based on the schemes developed by Niblack et al. (2002). The response of soybean genotypes to SCN populations was evaluated based on the method developed by Schmitt and Shannon (1992). Plants were classified as resistant (FI < 10), moderately resistant (FI = 10-30), moderately susceptible (FI = 31-60) or susceptible (FI > 60).

Data were subjected to analysis of variance using SPSS One-Way ANOVA (IBM, Armonk, New York, USA). Tukey’s HSD test (P < 0.05) was used to evaluate the influence of difference.

After more than five generations of single-cyst culture, only two cultures from Anda soil sample (Anda1 and Anda2) and one from Wuchang were available to be used for HG Type identification. The other cultures with no reproduction were discarded. SCN reproduction from single-cyst cultures of three SCN populations on the indicator lines, ‘Pickett’ and the susceptible check ‘Lee 68’ are shown in Table 2. The Anda1 culture was identified as HG Type 1.2.3.5.6.7, HG Type 1.3 for Anda2, and HG Type 2.5.7 for Wuchang (Table 2).

Significant differences (P < 0.05) occurred in number of cysts among soybean lines with each individual population (Tables 3-5). Based on female indices and resistance classification (Schmitt and Shannon, 1992), none of the 18 tested soybean cultivars/germplasms showed resistance to any of the three populations. ‘Kangxian12’ and ‘Kangxian13’ showed resistance to HG Type 2.5.7 in both tests (Table 4) and moderate resistance to both HG Type 1.2.3.5.6.7 and HG Type 1.3 in one or two tests (Tables 3 and 5). One breeding line ‘09-138’ showed resistance to HG Type 1.3 (Table 3) and susceptibility to HG Type 2.5.7 (Table 4) in both tests. Breeding line ‘09-138’ showed resistance to HG Type 1.2.3.5.6.7 in test 1 but moderate susceptibility in test 2.

Out of eleven local cultivars originally resistant to HG Type 0-, at least nine cultivars were identified as resistant to HG Type 2.5.7 in both tests (Table 4). All other genotypes was moderately resistant, moderately susceptible or susceptible to the three HG Types (Tables 3-5). Within susceptible genotypes, wide variation of nematode reproduction was observed (Tables 3-5). For example, the range of female indices was 60 to 152 for HG Type 1.3, 71 to 146 for HG Type 2.5.7 and 66 to 213 for HG Type 1.2.3.5.6.7 (Tables 3-5).

Among all tested soybean genotypes, the correlation between egg number (data not shown) and cyst number per plant was 0.7247 (test 1) and 0.6560 (test 2) for HG Type1.3, 0.9474 (test 1) and 0.9774 (test 2) for HG Type2.5.7, and 0.6679 (test 1) and 0.7632 (test 2) for HG Type1.2.3.5.6.7.