ZHENG Na, ZHANG Liu-ping, GE Feng-yong, HUANG Wen-kun, KONG Ling-an, PENG De-liang,LlU Shi-ming,

1 Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

2 College of Plant Protection, Hunan Agriculturaluniversity, Changsha 410128, P.R.China

Abstract Fusarium is usually thought to cause soybean root rot, which results in a large quantity of annual yield loss in soybean production, by its secretions including Fusarium toxins and cell wall degrading enzymes, but not by the conidia themselves that do not underlie any virulence so far. Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone. We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis. Of them, except for one isolate that showed diversity of virulence to different soybeans (virulent to one soybean whereas avirulent to another soybean), the others were all virulent to the two tested soybeans: both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection, and their virulence had dosage effects that only conidia cultures of at least 5×106 conidia mL-1 could show virulence to soybean; however, the sole conidia of the F. solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted. Finally, we developed the specific cleaved amplified polymorphic sequences(CAPS) markers to easily differentiate Fusarium isolates. The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of fusarium to soybean.

Keywords: Fusarium, soybean root rot, conidia, secretions, virulence, cleaved amplified polymorphic sequences (CAPS)marker

1. lntroduction

Soybean (Glycine max (L.) Merr.) is one important crop in the world, providing a sustainable source of protein and oil. During the growth and development, soybean is attacked by many kinds of pathogens worldwide causing huge yield losses annually as wellas making soybean quality considerably affected. We developed an ethane methylsulfonate-mutagenesis population of soybean PI 437654 as the new genetic soybean resources (data not yet published). From this population, we found 11 M2(the second generation mutants) plants of random spotdistribution 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 wallof 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 hostspecific, 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 offungus 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 offungi. Kwon and Anderson (2002) developed the restriction fragment length polymorphism (RFLP) makers with enzymatic digestion of EcoRV, PvuII and AvaI 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 byfiltering)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 Sciences by our lab. The wilted plants were collected from the plants, which were derived from the M2 seeds of the chemical mutagenesis PI 437654 population mentioned above, and planted in the samefield. Totally, 11 wilted soybean plants were acquired after growning in the field for about 2 months.

2.2. lsolation offungi from the wilted plants

The fungi were isolated from those 11 wilted plants collected using the method described by Fang (1998) and Qiu et al.(2011) with some modifications. In brief, the plants were rinsed and cleaned by tap-water, and then disinfected with 2% NaClO for 10 min, followed by 2-3 times of washing with 75% EtOH and sterilized ddH2O. Afterwards, a short stem of about 1 cm at the disease and health junction part of each plant was cut using an aseptic blade and inoculated and cultured on a PDA plate with 100 μg mL-1ampicillin at 25°C. After 3 d, the single colony was rinsed from the agar surface using sterilized ddH2O and the formed suspension was observed under an inverted fluorescence microscope(Olympus IX71, Japan). The suspension wasfinally diluted to 3-5 conidia in a 10-fold microscope vision using sterilized ddH2O. Subsequently, a little conidia suspension was cultured on a 2% agar plate at 25°C for 2 h, and then the conidia with germ tube emerging were selected and cultured on a new PDA plate with 100 μg mL-1ampicillin at 25°C.Each new single colony was taken as an isolate.

2.3. Observation of morphology of isolated fungi

The mycelia, conidiophores, conidia, microconidia,macroconidia, and chlamydospores of the fungi isolated were observed under an inverted fluorescence microscope(Olympus IX71, Japan).

2.4. Alignment and haplotype detection of lTS sequences of isolated fungi

The ITS sequences of all the isolated fungi were PCR-amplified using the specific primers for Fusarium spp. (forward:5´-CTTGGTCATTTTAGAGGAAGTAA-3´ (Gardes and Bruns 1993), and reverse: 5´-TCCTCCGCTTATTGATATGC-3´(White et al. 1990)). The PCR amplification was performed with the following program: denatured at 94°C for 4 min, 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min,and extended at 72°C for 7 min. The PCR products were separated on a 1.5% agarose gel, the size-corrected band was then cut and recovered and purified using AxyPrep DNA Gel Extraction Kit (Axygen, USA). The purified band was subsequently ligated into pMD19-T vector (TaKaRa,Japan) at 16°C overnight and the constructed plasmids were transformed into competent cells Escherichia coli DH5α (TaKaRa, Japan) using heat shock treatment method.The transformed E. coli was recovered and grown on the LB plate with IPTG and X-galat 37°C overnight. In the second day, the white colonies were selected and grown in liquid LB medium. Afterwards, the plasmids were extracted from the cultures using EasyPure Plasmid MiniPrep Kit(Transgen Biotech, China). Finally, the extracted plasmids were sequenced at Sangon Biotech Co. (Beijing, China)to gain the ITS sequences. The obtained ITS sequences were aligned, and the haplotypes were detected within the same group.

2.5. Construction of a phylogenetic tree of fusarium isolates

While blasting the obtained ITS sequences, the sequences of high similarity were selected. The phylogenetic tree was constructed using the ITS sequences of fusarium isolates and the selected sequences with high similarity by Neighbor-Joining method employing Mega 6.06 software.

2.6. Virulence of fusarium isolates to soybeans

The Fusarium isolates were cultured in liquid PDA medium at 25°C, 200 r min-1. The conidia were counted under an inverted fluorescence microscope (Olympus IX71, Japan)after mycelia were 4-layer gauze-filtered from the cultures cultured for about 7-9 days. The cultures after removalof mycelia (conidia cultures) werefinally diluted to be equal to 1×107conidia mL-1or to the concentration as needed, and then about 50 mlof conidia cultures, supernatants after centrifugation of cultures (secretions) or conidia suspended with ddH2O were loaded in a plastic open cup. Four tofive soybean seedlings that were of almost similar height at the growth stage with two true leaves completely unfolded were roots-immersed in the conidia cultures, secretions or conidia suspension in plastic cup and grew at 25°C. The virulence of fusarium isolates were observed and recorded each day,totally about 5-7 days.

2.7. Development of CAPS markers

Regarding the discrimination of these four groups of fusarium isolated,first, according to the ITS sequences amplified, the amplicon off. solani is remarkably longer than that of the other three Fusarium groups, which was easily distinguished from the other groups by direct separation on the 3% agarose gel. Then, on the basis of the ITS sequences of the other three groups, we developed the CAPS markers by the digestion of the amplified ITS sequences as mentioned above using combined enzymes of AvaI, SptI and BspCNI (New England BioLabs, USA).The enzymatic digestion was performed by the instruction of manufacture.

3. Results

3.1. lsolation and morphology of fusarium spp. from the wilted plants in a soybean-production field

Fig. 1 Morphological features of Fusarium isolated from the wilted plants in a soybean-production field. A, E, I and L, mycelia. B, F, J and M, conidiophores. C, conidia. D,chlamydospores. G, microconidia. H, macroconidia. K and N, both microconida and macroconidia. The bar in the pictures denotes 50 μm of length.

Totally, we isolated 18 fungi from the wilted plants of a soybean-production field in China, and these fungi showed different morphologies. According to the results offollowing molecular identification, one isolate (#1) is a Frametes hirsute (data not shown), completely different from the other 17 isolates, hereby we just discuss the other 17 isolates,from which we observed 4 main types of morphologies(Fig. 1). One isolate #11, villiform aerial mycelia appeared in white after cultured on PDA plate for 8 days; conidiophores were short; all conidia were large (macroconidia) with 2 or 3 diaphragms, sickle-shaped, and both ends became narrow gradually, no small conidia (microconidia)were observed; and it had round chlamydospores with yellowish-brown spore-wall, which emerged on top of or among mycelia (Fig. 1-A-D). One isolate #15, cottony or arachnoid mycelia were long; almost allof mycelia were in white but some were in purplish-red; conidia emerged on conidiophores and consisted of microconidia and macroconidia, but the quantity of microconidia was significantly more than that of macroconidia, microconidia were in ovalor round with no diaphragms observed,while macroconidia were in sickle-shaped with obviously narrowed ends and 3-5 diaphragms; no chlamydospores were observed, (Fig. 1-E-H). One isolate #4, cottony aerial mycelia were in white and short; conidia emerged on conidiophores, and were almost microconidia with a small proportion of macroconidia, microconidia were in ovalor kidney-shaped with no diaphragms or just one diaphragm observed, whereas macroconidia were in sickle-shaped, nearly straight or slightly bended with a little narrowed or blunted ends, and had 2-5 diaphragms, usually 3 diaphragms; no chlamydospores were observed (Fig. 1-IK). The other isolate #12, thick and villiform aerial mycelia were in white; cylinder-shaped conidiophores were short;conidia were the mixture of microconidia and macroconidia,of which microconidia were in round, oval, or kidneyshaped with 0-3 diaphragms, while macroconidia were in sickle-shaped, slightly bended, both ends became narrow,and 3-7 diaphragms were observed in macroconidia,no chlamydospores were observed (Fig. 1-L-N). In combination with the following molecular identification, all those 17 isolated fungi are identified as Fusarium spp.

3.2. Molecular identification of the isolated Fusarium spp.

We continuously performed the molecular identification offungi isolated. First, we obtained the ITS sequences of isolates by PCR amplification using the known specific ITS primers for Fusarium spp. (White et al. 1990; Gardes and Bruns 1993). We aligned the ITS sequences and clearly classified them into 4 groups, and within the appropriate groups, isolates #2, #7, #15, and #17 were detected to show several haplotypes, having one deletion, two single nucleotide polymorphisms (SNPs), one SNP, and two insertions and one SNP at different positions, respectively (Fig. 2).

Subsequently, we blasted all the ITS sequences on NCBI website (http://www.ncbi.nlm.nih.gov). All the 17 isolates belong to Fusarium spp. With all the sequences of high similarity selected, a phylogenetic tree was constructed using Mega 6.06 software with Neighbor-Joining method(Fig. 3). The 17 isolates are all Fusarium spp. and can be clustered into 4 groups similar to the alignment results:Isolates #2, #3, #5, #8, #9, #14, #15, and #17 are classified in the F. oxysporum group; isolate #4 is a F. solani; isolates#6, #7, #10, #11, #16 and #18 are classified in the F. equiseti group; and isolates #12 and #13 are in the F. commune group. Thus, the 17 isolates in this work are all Fusarium spp. including F. oxysporum, F. equiseti, F. solani, and F. commune.

Fig. 2 Alignment and haplotype detection of the amplified specific internal transcribed spacer (ITS) sequences of the Fusarium spp. isolated from the wilted soybean plants. The alignment was conducted using the amplified ITS sequences of 17 isolates,showing 4 groups: The 1st includes 6 isolates (#6, #7, #10, #11, #16, and #18), the 2nd contains 8 isolates (#2, #3, #5, #8, #9,#14, #15, and #17), the 3rd comprises of 2 isolates (#12, and #13), and the 4th has just 1 isolate (#4). Within the corresponding groups, isolates # 2, #7, #15, and #17 were detected to show haplotypes, having 1 deletion, 2 single nucleotide polymorphisms(SNPs), 1 SNP, and 2 insertions and 1 SNP at different positions, respectively.

3.3. Virulence of fusarium isolates to soybean

Fig. 3 Phylogenetic analysis of the isolates using the amplified specific internal transcribed spacer (ITS) sequences. By blasting the amplified ITS sequences of the isolates the sequences with high similarity were selected. The phylogenetic tree was constructed using the Mega 6.06 software with Neighbor-Joining method. Phylogenetic analysis clearly indicates that the 17 isolates were able to be clustered into 4 Fusarium groups: 8 F. oxysporum isolates (#2, #3, #5, #8, #9, #14, #15, and #17), 1 F. solani isolate (#4), 6 F. equiseti isolates (#6, #7, #10, #11, #16, and #18), and 2 F. commune isolates (#12 and #13).

We used the conidia cultures of each isolate to infect the about 10-day-old seedlings of soybeans. The cultures were used at the concentration of 1×107conidia mL-1, and the soybeans PI 437654 and Zhonghuang 13 were infected.We observed the virulence to soybean seedlings each day post infection totally for 5-7 days. The observation indicates that if the isolates were virulent to soybean seedlings,some brown spots would begin to appear at the stem base(Fig. 4-A), and the seedlings would start to wilting at 2 or 3 days post infection, dependent on the virulence degrees; the seedlings would usually be wilted at 5 days post infection(Fig. 4-B and C). After 5 days post infection, the phenotype of seedlings would not be changed much. The virulence of 17 isolates to soybeans are summarized in Fig. 4-L,indicating almost of isolates were virulent to soybean seedlings, except the isolate #18, which was also virulent to Zhonghuang 13, but avirulent to PI 437654 (Fig. 4-D-K).

Successively, we tested the dosage effects of conidia cultures of isolates on the virulence to soybean seedlings.The results indicate that the cultures at concentration of 5×106conidia mL-1could make seedlings wilted at 5 days post infection, but the dilute of conidia cultures (at 1×106,5×105, and 1×105conidia mL-1) did not (Fig. 5). Taken to gether with the results mentioned above that the cultures at concentration of 1×107conidia mL-1caused the seedlings wilted at 5 days post infection (Fig. 4), it can be concluded that the virulence offusarim to soybean requires the dosage of conidia that the concentration of conidia cultures should

Fig. 4 Virulence of Fusarium spp. isolated from soybeans. A, symptoms of roots and stem base at 5 days post infection with (left)and without (right) infection of Fusarium, respectively. B and C, wilting of seedlings at different degrees at 5 days post infection.D-K, virulence of isolate #18 to soybeans. D-G, PI 437654. H-K, Zhonghuang 13. D, F, H and J, CK. E, G, I and K, infection of conidia cultures of isolate #18. D, E, H and I, 0 day post infection. F, G, J and K, 5 days post infection. L, summary of the virulence of Fusarium spp. isolated to soybean. The conidia culture at the concentration of 1×107 conidia mL-1 was used. The Fusarium species is virulent (Vir) to soybean if the soybean seedlings were wilted, while the Fusarium species is avirulent (Avir)to soybean if the soybean seedlings were not wilted, at 5 days post infection.

Fig. 5 Determination of effective concentration of conidia cultures of Fusarium spp. causing wilting of soybean seedlings. Taken to gether with the results that the conidia cultures at the concentration of 1×107 conidia mL-1 caused the wilting of seedlings (Fig.4), the results show that the conidia cultures at the concentration of equal to or over 5×106 conidia mL-1 caused the wilting of seedlings. A-E, 0 day post infection. F-J, 5 days post infection. A and F, CK. B and G, C and H, D and I, and E and J, conidia cultures at the concentration of 5×106, 1×106, 5×105, and 1×105 conidia mL-1, respectively. F. equiseti isolate #11 was used to infect soybean Zhonghuang 13.

b

e equal to or over 5×106conidia mL-1.

3.4 Conidia of an isolated F. solani alone are able to be virulent to soybean

Fig. 6 The representative virulence phenotypes of the secretions or conidia of Fusarium spp. to soybean. Cultures of isolate #11 at the concentration of 1×107 conidia mL-1 werefiltered to remove mycelia and gain conidia cultures, and then conidia cultures were centrifuged and separated into supernatants as the secretions and debris. The obtained debris was washed with sterilized ddH2O twice and centrifuged as the conidia, and then the conidia were suspended with appropriate volume of ddH2O as the conidia solution. A-H, PI 437654. I-P, Zhonghuang 13. A, E, I and M, CK. B, F, J and N, conidia cultures. C, G, K and O, supernatants.D, H, land P, conidia. A-D and I-L, 0 day post infection. E-H and M-P, 5 days post infection.

Fig. 7 Conidia of Fusarium solani isolate #4 alone underlies virulence to soybeans. Of the 17 isolated Fusarium spp., the isolate#4 at the concentration of 1×107 conidia mL-1 showed that the conidia caused the wilting of seedlings of PI 437654 and Zhonghuang 13, whereas the others did not. Cultures of isolate #4 at the concentration of 1×107 conidia mL-1 werefiltered to remove mycelia and gain conidia cultures, and then conidia cultures were centrifuged and separated into supernatants as the secretions and debris. The obtained debris was washed with sterilized ddH2O twice and centrifuged as the conidia, and then the conidia were suspended with appropriate volume of ddH2O as the conidia solution. A-H, PI 437654. I-P, Zhonghuang 13. A, E, I and M, CK.B, F, J and N, conidia cultures. C, G, K and O, supernatants. D, H, land P, conidia. A-D and I-L, 0 day post infection. E-H and M-P, 5 days post infection.

To our best knowledge, the virulence of fusarium to plants is usually caused by the secreted cell degrading enzymes(Cooper et al. 1998; Jayasinghe et al. 2004; Paccanaro et al.2017) and toxins such as fusaric acid, FvTox1, T-2 toxin,and endo-xylarases (Brito et al. 2006; Brar et al. 2011),and no studies were reported that conidia of fusarium underlies virulence to plants alone so far. Truly, for almost allof 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 1×107conidia 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 5×106conidia mL-1to soybean. The results showed that the conidia at the concentration of 5×106conidia mL-1did 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 1×107conidia mL-1.

3.5. Development of CAPS markers to differentiate Fusarium spp.

According to the ITS sequences amplified, the amplicon off. solani is obviously longer than that of the others (Fig. 2),so wefirstly 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 BspCNI with AvaI, PstI 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 allof 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 thatfield 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.

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.

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 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 5×106conidia mL-1can 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 1×107conidia mL-1is required for the isolate #4 conidia to show the virulence to soybean (data not shown). Obviously,it is a novelfinding. 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 allof 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 wellas 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, wefirst 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 off. 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 BspCNI combining AvaI, PstI 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 F. commune; secondly, besides secretions, conidia of one F. solani isolate #4 alone exhibited virulence to soybean and could make soybean seedlings wilted, a newfinding that Fusarium conidia themselves can show virulence to soybean, this isolate #4 will likely be used to investigate the new mechanism of virulence of fusarium to soybean;and thirdly, the specific CAPS markers were developed to easily differentiate Fusarium spp.

Acknowledgements

This work wasfinancially supported by the Innovation Program and Youth Elite Program of Chinese Academy of Agricultural Sciences. We thank very much many other colleagues and students in our lab for their kind helps and assistance in the collection of soil samples in the field.