JIA-2018-09

2050 ZHENG Na et al. Journal of Integrative Agriculture 2018, 17(9): 2042–2053 all of Fusarium spp. isolated in this work, only the conidia cultures and secretions (supernatants after centrifuge of conidia cultures) were virulent to soybeans, both PI 437654 and Zhonghuang 13 (Fig. 6), meaning that in this work, the virulence of those Fusarium spp. to soybean is also caused by the secretions. However, we screened one F . solani isolate #4, not only whose conidia cultures (Fig. 7-F and N) and secretions (Fig. 7-G and O) but also whose conidia alone at the concentration of 1 × 10 7 conidia mL –1 (Fig. 7-H and P) were virulent to soybeans and could make soybean seedlings wilted, just the virulent strength of conidia was weaker than that of cultures and secretions (Fig. 7). Furthermore, the virulence of isolate #4, including its conidia cultures, secretions and conidia, to PI 437654 all seemed stronger than to Zhonghuang 13 (Fig. 7). Subsequently, we tested the virulence of the isolate #4 conidia at 5×10 6 conidia mL –1 to soybean. The results showed that the conidia at the concentration of 5×10 6 conidia mL –1 did not display any virulence to soybean seedlings of both PI 437654 and Zhonghuang 13 (data not shown). Thus, some dosage is required for the virulence of conidia of the isolate #4 to soybean, namely, at least 1×10 7 conidia mL –1 . 3.5. Development of CAPS markers to differentiate Fusarium spp. According to the ITS sequences amplified, the amplicon of F . solani is obviously longer than that of the others (Fig. 2), so we firstly distinguished F . solani from the other three Fusarium spp. by the direct separation of PCR products on a 3% agarose gel. There are polymorphisms between F . solani and the other three Fusarium spp. (Fig. 8-A). Afterwards, we analyzed and selected the digestion sites of restriction endoenzymes on the amplified ITS sequences, and developed the CAPS markers with the combination of Bsp CNI with Ava I, Pst I or both. Three Fusarium spp. show different digestion patterns in all the combinations, easily being discriminated (Fig. 8-B). 4. Discussion Because all those 11 wilted soybean plants used in this work are the mutants of PI 437654, which is resistant to almost all of SCN races (Wu et al . 2009), and SCN, a devastating pathogen in soybean production worldwide causing severe economic damage each year (Koenning and Wrather 2010), was previously surveyed in that field planted with PI 437654 mutants population (data not shown), we still counted SCN cysts in those wilted plants although the symptoms were those of soybean root rot disease. As a result, we could not observe any cysts in the roots of these wilted plants (data not shown), so, we could eliminate the possibility of SCN disease on them due to the mutation and then (partial) loss of the resistance to SCN. Totally, we isolated 18 fungi from the wilted plants of soybean PI 437654 mutants. Except for isolate #1 which belongs to Frametes hirsute identified by blasting using the ITS sequences amplified (data not shown), as expected, all the other 17 isolated fungi belong to Fusarium spp. identified through the phylogenetic analysis using amplified ITS sequences and the high similarity sequences blasted in NCBI (Fig. 3). The fungus Fusarium has a variety of species (Zakaria and Lockwood 1981; Zhang et al . 2010). According to the phylogenetic tree constructed, those Bsp CNI /Ava I Bsp CNI /Pst I Bsp CNI /Ava I /Pst I M #8 #11 #12 M #8 #11 #12 M #8 #11 #12 M M #11 #8 #4 #12 M A B Fig. 8 Development of cleaved amplified polymorphic sequences (CAPS) markers to distinguish Fusarium spp. Four isolates each from F. oxysporum , F. equiseti , F. solani , and F. commune were selected to be PCR-amplified using the specific internal transcribed spacer (ITS) primers listed in the Section of Materials and methods. A, separation patterns of ITS sequences amplicon on agarose gel. F. solani could be distinguished from the other three Fusarium spp. by the polymorphisms after separation on a 3% agarose gel. B, different patterns of enzymatic digestion of the PCR-amplified products of ITS sequence of Fusarium isolates. The PCR-amplified products were digested by the combined restriction endoenzymes. M is DNA ladder; #8, #11 and # 12 are F. oxysporum isolate #8, F. equiseti isolate #11 and F. commune isolate #12 isolated in this work, respectively.