JIA-2018-09

2051 ZHENG Na et al. Journal of Integrative Agriculture 2018, 17(9): 2042–2053 isolates in this work are clearly classified into 4 groups, including 6 F . equiseti isolates, 8 F . oxysporum isolates, 2 F . commune isolates, and 1 F . solani isolate (Figs. 2 and 3). From the ITS sequences amplified, some haplotypes were detected, the isolates #2, #7, #15, and #17 show 1 deletion, 2 SNPs, 1 SNP, and 2 insertions and 1 SNP at various positions, respectively, within the appropriate groups (Fig. 2). These 4 isolates are all virulent to soybeans similar to the other isolates except isolate #18 (Fig. 4-L), so all the detected haplotypes (insertions, deletions and SNPs) do not play a (key) role in the virulence to soybean in these isolates. We also observed 4 main types of morphologies of Fusarium including their mycelia, conidiophores, conidia, and chlamydospores in this work. We just observed the chlamydospores in the type of isolate #11 ( F . equiseti ), no chlamydospores were observed in the other three types of isolates (Fig. 1). On the other hand, they were different in the size and number of diaphragm of conidia, and the ratio of microconidia and macroconidia, some species majorly had small conidia (microconidia), some were only observed to have large conidia (macroconidia), while some had both (Fig. 1). For instance, the isolate #11 was only observed to show macroconidia (Fig. 1-C); but all the macroconidia observed were in sickle-shaped, so all the isolates are Fusarium spp., consistent with the results of molecular identification (Figs. 2 and 3). As we know, the fungus Fusarium such as F . graminearum , and F . virguliforme causes the diseases of plants by the secreted cell wall degrading enzymes and toxins (Islam et al . 2017; Paccanato et al . 2017). On the other hand, the fungus invades the roots of soybean, and then enters into vascular bundle and secrets a number of toxins which are translocated to the stem, making stem displaying symptoms such as water-soaking brown spots that subsequently become bigger and bigger and appear more and more, finally, the plants wilt (Bushnell et al . 2003; Brown et al . 2010). In the present work, we surveyed the virulence of the 17 isolated Fusarium spp. to soybeans PI 437654 and Zhonghuang 13. Except for the isolate #18, the conidia cultures and secretions of other isolates showed virulent to the soybeans (Figs. 4 and 6). That’s to say, the secretions of those Fusarium spp. caused the virulence to soybean in this work. If the Fusarium was virulent to soybean, after infection, brown spots appeared in the stem base (Fig. 4-A), and then the seedlings started to wilt at 2–3 days post infection (Fig. 4-B and C). All these results are consistent with the mentioned above. Moreover, the virulence of isolates to soybean showed dosage effects. While the species is virulent to soybean, only the conidia cultures at the concentration of at least 5×10 6 conidia mL –1 can make the seedlings wilted (Fig. 5). These results may also suggest, in our opinion, that over some quantity of the secreted toxins or cell wall degrading enzymes is required to produce the virulent effects to plants. To our best knowledge, conidia of Fusarium has not been reported to be virulent to plants. In this work, we isolated one F . solani isolate #4 (Fig. 3), not only whose secretions but also whose conidia were virulent to soybeans PI 437654 and Zhonghuang 13 and could make soybean seedlings wilted, although the symptoms of seedlings infected with conidia was weaker than those of seedlings infected with secretions (Fig. 7). Furthermore, the results from the test of dosage effects of conidia indicate that equal to and over 1×10 7 conidia mL –1 is required for the isolate #4 conidia to show the virulence to soybean (data not shown). Obviously, it is a novel finding. We suppose that the conidia of this species may produce some substances that interact with soybean, which are being investigated. Soybean SCN and root rot diseases are the top 2 diseases on soybean making huge damage to soybean. It is the best if genetic resources can be identified to underlie resistance to both pathogens and cultivated in the soybean production areas or used for breeding. PI 437654 is resistant to almost all of SCN races (Wu et al . 2009), while Zhonghuang 13 is susceptible to SCN (data not shown). In this work, the Fusarium isolate #18 is avirulent to PI 437654 but virulent to Zhonghuang 13 (Fig. 4-D–K), so actually PI 437654 can be used as a Fusarium -resistant as well as SCN-resistant source to map and identify the resistant gene(s) and then for resistance breeding. The highly conserved ITS sequences are one marker for the fungi (Schoch et al . 2012), and the ITS sequences of various species within the same genera show some differences, so we can utilize the ITS sequences to develop the molecular markers for the discrimination of the Fusarium spp. (Del-Prado et al . 2010; Schoch et al . 2012). In this work, we first distinguished F . solani from the other three Fusarium spp. ( F . oxysporum , F . equiseti , and F . commune ) directly by the agarose gel separation using the PCR- amplified ITS products (Fig. 8-A) because the ITS sequence of F . solani at the amplified region is much longer than the others (Fig. 2). Subsequently, we developed the CAPS markers to differentiate the three Fusarium spp. by the enzymatic digestion. These three species could be clearly discerned after digestion by Bsp CNI combining Ava I, Pst I or both (Fig. 8-B). These markers can be used to more quickly identify Fusarium spp. than the survey of virulence to the hosts. 5. Conclusion In the present work, firstly, 17 Fusarium isolates were isolated and molecularly identified from a soybean-production field, which distribute in F . equiseti , F . oxysporum , F . solani , and