JIA-2019-11

2564 LIU Ying et al. Journal of Integrative Agriculture 2019, 18(11): 2561–2570 2.4. Biological control effect of A. welwitschiae in the greenhouse One day before inoculation of J2 nematodes, roots of 3-week- old rice plants were drenched with 5 mL liquid PDB medium or conidial suspension at a concentration of 10 6 conidia mL –1 . Then, 300 J2s of M . graminicola were inoculated around the roots of each plant. Each treatment was replicated eight times, and the treatments were arranged in a randomized complete block design. After incubation in the greenhouse for 14 d, the root galls in each rice plant were counted directly with the naked eye. Nematodes in rice roots were counted under a stereomicroscope after staining as described above. After distaining in 4% acidified glycerol for 3–4 d, nematodes inside the roots at each developmental stage were counted under a stereomicroscope. To calculate the ratio of nematodes and analyze the effect of A . welwitschiae on nematode development, the number of nematodes in different life stages (female or third/fourth-stage juvenile (J3/J4)) was divided by the total number of nematodes in roots using Microsoft Excel 6.0 (Redmond, Washington, USA) (Zhan et al . 2018). 2.5. Statistical analysis All data were subjected to ANOVA using SAS Software version 8.0 (SAS Institute, Cary, NC, US). Significant differences ( P ≤0.05) between the treatments were determined according to Duncan’s multiple range test. 3. Results 3.1. Identification of A. welwitschiae Morphological characteristics of fungal isolates were assessed according to Raper and Fennell (1965). Colonies grewrapidlyonCYAat 25°C, reachingadiameter of 43–45mm in 5 d; at conidiogenesis, dense, brown-black; mycelia white with faint yellow; exudate and soluble pigment absent; reverse yellowish white. Conidial heads small, spheroidal, 200–400 μm in diameter; conidiophores borne from aerial hyphae,stipessmooth-walled,brownincolor,(1000–3000)µm× (10–18) µm; vesicles spheroidal, 40–60 µm in diameter, biseriate; metulae short, (10–20) µm×(5–7) µm; phialides ampuliform, (8–10) µm×(2–3) µm; conidia spheroidal, conspicuously roughened, 4–6 µm (Fig. 1). Molecular identi cation of fungal isolates was con rmed by sequencing of BenAand CaM, and the sequencing results were deposited in GenBank under accession numbers MH021601 and MH021602, respectively. Evolutionary history was inferred using the NJ method with Mega 3 Software (Kumar et al . 2004). The partial BenA data set consisted of 556 characters, and phylogenetic analysis showed that the genetic similarity between the tested fungal isolate and A . welwitschiae CBS 139.54 (FJ629291.1) was 89% (Fig. 2-A). The partial CaM data set consisted of 616 characters, and the dendrogram constructed from the sequence confirmed that the genetic similarity between CaM of the tested fungal isolate and A . welwitschiae CBS 139.54 (KC480196.1) was 75% (Fig. 2-B). The morphological characteristics and sequence similarity results confirmed that the fungal isolate we collected belongs to A . welwitschiae . 3.2. A. welwitschiae has toxic effects on the behavior of M. graminicola The toxicity of A . welwitschiae to eggs and J2s of M . graminicola was tested in vitro in two separateexperiments. The ovicidal potential of A . welwitschiae against egg hatching of M . graminicola is shown in Fig. 3-A. The conidial suspension of AW2017 significantly reduced nematode egg hatching compared with the PDB control at 4 and 8 d of incubation. After incubation for 4 d, the percentage of egg hatching in the 5× and 10×AW2017 treatments was (35.7±3.5)% and (22.0±3.3)% lower than that in the control treatment, respectively. After incubation for 8 d, the percentage of egg hatching in the 5× and 10×AW2017 treatments was (47.3±4.9)% and (39.4±4.3)% lower than that in the control treatment, respectively. The larvicidal potential of A . welwitschiae against the J2 individuals of M . graminicola is summarized in Fig. 3-B. After incubation in the conidial suspension of A . welwitschiae for 48 and 72 h, all conidial suspensions in different concentrations exhibited significant larvicidal potential. After incubation for 48 h, the highest percentage of juvenile mortality was recorded at the concentration of 5×AW2017, followed by the concentration of 10×AW2017, which caused mortality rates of (63.8±5.8)% and (40.4±6.4)%, respectively. The lowest percentage of juvenile mortality was recorded in the PDB control treatment, which caused only (9.8±3.2)% mortality to J2s and was significantly different from the AW2017 treatment. Similar results were also observed after A B C Fig. 1 Morphological characteristics of isolates of Aspergillus welwitschiae AW2017. A, colonies on CYA after 5 d at 25°C. B, conidiophores (Bar=10 µm). C, conidia (Bar=5 µm).