JIA-2019-11

2562 LIU Ying et al. Journal of Integrative Agriculture 2019, 18(11): 2561–2570 Southeast Asia, where approximately 90% of the world’s rice is grown and consumed (Padgham and Sikora 2007; Kyndt et al . 2012; Ji et al . 2013). After second-stage juveniles (J2s) invade the root tips, they inject pharyngeal secretions into vascular cells to induce a specialized feeding site that is then used as a food resource throughout their life cycle. M . graminicola causes terminal, hook-shaped galls, which are characteristic symptoms of infection by this nematode species (Khan et al . 2012; Kyndt et al . 2013). Field experiments in different countries have indicated that M . graminicola can cause yield losses of between 16 and 87% (Soriano and Reversat 2003; Padgham et al . 2004; Prasad et al . 2010; Dutta et al . 2012). In India, this nematode has been found to cause yield losses of 16 to 32% in rain-fed and upland rice (Prasad et al . 2010). In the Philippines, treatment with carbofuran resulted in a 34% increase in rice yield. Nematicide application in upland rice fields in Indonesia resulted in yield increases of 28 to 87% (Dutta et al . 2012). The ability of M . graminicola to survive long periods in anoxic environments and rapidly re-infect rice roots in the absence of flooded conditions has led to its widespread distribution from completely aerobic upland systems to lowland rain-fed and deep-water rice production systems (Padgham et al . 2004; Bridge et al . 2005; Padgham and Sikora 2007). Several studies have indicated that various nematode management methods are effective in controlling rice RKNs (Gaur and Pankai 2010). Despite the deleterious effects of chemicals on humans and the environment, the application of nematicides (e.g., organophosphates, carbamates and chlorpyriphos) is still the most effective means of nematode management in rice ecosystems (Khan et al . 2012). The application of carbosulfan and chlorpyriphos to rice seedlings through root-dipping treatment was found to significantly suppress root galls and increase plant growth. Soil application of phorate and fosthiazate also significantly reduces galling on rice plants (Prasad and Rao 1977; Rui et al . 2015). However, chemical nematicides are not economically feasible due to the low market value of rice (Mantelin et al . 2017). In the case of floodwater management, the recent adoption of labor- and water-conserving practices, e.g., direct seeding and furrow irrigation, could potentially increase infection by M . graminicola . The lack of nematode-resistant rice cultivars and the economic unviability of nematicide application severely limit options for effective nematode management. Therefore, the development of alternative strategies that combine resistance breeding with biological control is needed. Biological control using endophytic microorganisms has been demonstrated to be effective at reducing the penetration, delaying the development and diminishing the reproductive capacity of sedentary and migratory parasitic nematodes (e.g., Meloidogyne incognita on tomato and Radopholus similis on banana) (Hallmann and Sikora 1994; Vu et al . 2006; Menjivar et al . 2011; Martinuz et al . 2013; Le et al . 2016). Despite M . graminicola ’s widespread occurrence in rice production systems, only limited research has been performed on its biological control. Le et al . (2009) showed that root-galling severity was reduced by 38% with treatment by the rhizosphere fungi Trichoderma spp. Le et al . (2016) found that endophytic Fusarium moniliforme colonization significantly reduced M . graminicola penetration by 55% and delayed juvenile development into females inside rice roots. Treatment prior to invasion with Bacillus megaterium resulted in a nearly 60% reduction in juveniles migrating to the root zone and a greater than 40% reduction in nematode penetration (Padgham and Sikora 2007). Application of the nematode-trapping fungus Arthrobotrys oligospora to soil reduced the number of root knots by 57.6–62.0% and increased the fresh weight of roots by 38.9–44.2% (Singh et al . 2012). Co-inoculation of the nematode-trapping fungus Dactylaria brochopaga and the endoparasitic fungus Catenaria anguillulae significantly reduced the number of root knots of M . graminicola and increased the yield of rice plants (Singh et al . 2013). However, no evidence exists for the biological potential of Aspergillus welwitschiae against RKNs. To investigate the biocontrol efficacy of A . welwitschiae , the present investigation aimed to demonstrate: (i) the potential of A . welwitschiae to kill M . graminicola in vitro , and (ii) the effects of A . welwitschiae on the behavior, infection and development of M . graminicola in the greenhouse. 2. Materials and methods 2.1. Isolation and identification of A. welwitschiae The A . welwitschiae strain AW2017 was isolated from rhizospheric soil samples and stored at –80°C in the China General Microbiological Culture Collection Center, Beijing (CGMCC No. 14132). Amonoconidial culture of the isolate was prepared using material from colonies grown on Czapek yeast autolysate agar (CYA) at 25°C for 5 d and mounted in lactophenol without dye. Optical microscopic examination and photography were performed with a Nikon Eclipse 80i and Nikon Digital Sight DS-L1 microscope (Nikon Corporation, Tokyo, Japan) as per the method of Susca et al . (2016) and Raper and Fennell (1965). The resulting liquid cultures were incubated at 28°C in the dark with shaking (150 r min –1 ) for 5 d. Mycelia were filtered, lyophilized and ground for DNA extraction. DNA was then prepared with a Fungus Genomic DNA Extraction Kit (Solarbio Life Sciences, Beijing, China) according to