JIA-2018-09

2043 ZHENG Na et al. Journal of Integrative Agriculture 2018, 17(9): 2042–2053 (the second generation mutants) plants of random spot- distribution in the field started to wilt after planting of seeds in the field for about 2 months, and then analyzed them in detail in this work. That wilting is the symptoms of soybean root rot, a soil-borne disease majorly caused by the fungi Phytophthora sojae , Fusarium spp., Pythium spp. and Rhizoctonia solani (Naito et al . 1993; Nelson et al . 1997) that is increasingly more difficulty to be managed. Various pathogens cause different soybean root rot diseases, for example, P . sojae causes Phytophthora root rot (PRR), and Fusarium spp. such as F . virguliforme which can also lead to soybean sudden death syndrome (SDS) (Lightfoot 2015), F . oxysporum , F . solani , and F . equiseti give rise to Fusarium root rot (FRR). It is of priority to understand the nature of virulence of these pathogens and identify and utilize the resistant genetic soybean sources to effectively control them. Many virulent effectors of P . sojae and a number of quantitative trait loci (QTL) (genes) in soybean underlying resistance to P . sojae were mapped (Liu et al . 2016). More promisingly, recently, a new mechanism of virulence of P . sojae to soybean was reported that for the infection and virulence, P . sojae secrets PsXLP1, a paralogous PsXEG1, as a bait to bind more tightly with the conserved soybean apoplastic glucanase inhibitor protein GmGIP1 so that GmGIP1 has no opportunities to bind with PsXEG1 to lose the virulence (Ma et al . 2017). Some virulent genes of Fusarium associated with plant root rot disease were also identified (Bai et al . 2001; Islam et al . 2017). The virulence of Fusarium to soybean has been thought to be preliminarily triggered by the proteases and toxins secreted. The proteases, mainly the cell wall degrading enzymes including pectinase, cellulose and β-glucosidase, are secreted to degrade cell wall of hosts, allowing pathogens to possibly invade into host cells (Cooper et al . 1998; Jayasinghe et al . 2004; Paccanaro et al . 2017). Meanwhile, a number of toxins are secreted by the pathogens, these toxins bind with the cell membrane, and subsequently the structure of cell membrane of host is destroyed. Many kinds of toxins such as fusaric acid, FvTox1, T-2 toxin, and endo-xylarases were identified (Brito et al . 2006; Brar et al . 2011). These toxins are not host- specific, and they can be translocated from infected roots to shoots, making plants wilted (Tai et al . 2006). However, Fusarium conidia alone have not yet been reported to possess virulence to soybean, to our best knowledge. In China, much work has been performed in survey of Fusarium spp. In the soybean production areas, F . solani , F . equiseti , and F . oxysporum were predominantly surveyed in Shanxi province, China (Liu et al . 1992), and F . solani was majorly distributed in Fujian and Shandong provinces, China (Wu et al . 2008; Cui et al . 2010; Pan et al . 2010), while F . equiseti was mainly distributed in Heilongjiang Province (Zhang et al . 2010). The traditional methods to identify the pathogens through observation of morphological features and survey of their virulence to hosts are time-consuming and sometimes even inaccurate. The internal transcribed spacer (ITS) sequence of ribosome of fungus is highly conserved, which has been broadly used to quickly identify fungus in combination with observation of morphology (Guadet et al . 1989; Bruns et al . 2003). However, within the same genera, just little difference in the sequences of ITS regions exits among various species (Tooley et al . 1996). Therefore, the method utilizing restriction endoenzymes to digest the ITS sequences was developed to form the polymorphisms by the gel separation for the discrimination and identification of fungi. Kwon and Anderson (2002) developed the restriction fragment length polymorphism (RFLP) makers with enzymatic digestion of Eco RV, Pvu II and Ava I to analyze the relationships among wheat Fusarium spp. It is worthy of developing effective molecular markers to simply and quickly survey and identify Fusarium spp. In this work, we isolated the fungi from the wilted soybean plants, afterwards, molecularly identified the fungus species by phylogenetic analysis using the amplified ITS sequences specific for Fusarium , finding that except one isolate all the other fungi isolated belong to Fusarium . We subsequently tested the virulence of those Fusarium isolates to two soybean lines, PI 437654 and cultivar ( cv .) Zhonghuang 13, indicating most of those isolates, both its conidia cultures (cultures after mycelia removed by filtering) and secretions (supernatants after centrifugation of conidia cultures), were virulent to soybeans, more importantly, we screened a F . solani isolate #4 whose conidia alone were also able to make soybean seedlings wilted. Furthermore, our results indicate that the virulence of Fusarium to soybean had dosage effects. In addition, we developed cleaved amplified polymorphic sequences (CAPS) markers to easily differentiate the Fusarium spp. isolated. 2. Materials and methods 2.1. Soybeans and wilted plants Soybean PI 437654 and cv . Zhonghuang 13 were used as the materials in the present work. PI 437654 is a soybean genetic source underlying a broad resistance to almost soybean cyst nematode (SCN) races (Wu et al . 2009), whereas cv . Zhonghuang 13 is a soybean cultivar in China, which is susceptible to SCN, but having some good agronomy traits such as strong resistance to lodging. The chemical mutagenesis PI 437654 mutant population was developed at the Langfang Experimental Base of Institute of Plant Protection of Chinese Academy of Agricultural