Pathogenicity and Genetic Diversity of Fusarium oxysporum Causing Soybean Root Rot in Northeast China

Soybean is an important edible legume cultivated around the world. However, soybean production is seriously impacted by the widespread of root rot disease. In this study, genetic diversity and pathogenicity of Fusarium oxysporum associated with root rot of soybean in Heilongjiang province, China, were examined. A total of 50 F. oxysporum strains were isolated from diseased soybean plants grown in Harbin, Heihe, Jixi, Jiamusi and Qiqihar of Heilongjiang province. Pathogenicity study indicated that all F. oxysporum strains were able to induce root rot disease on soybean in which 28% of the isolates were highly aggressive, 42% were moderately aggressive, and 30% were weakly aggressive. Aggressiveness of the isolates did not appear to be associated with geographic location or plant age of isolation. Genomic DNA of the isolates was analyzed by polymerase chain reaction using eight amplified fragment length polymorphism (AFLP) primers that generated 1728 bands, of which 99% were polymorphic. Cluster analysis using UPGMA showed that the similarity values ranged from 0.15 to 0.47. At a similarity coefficient of 0.2, the isolates were separated into 7 groups. Analysis of molecular variance indicated that about 92% of the genetic variation resided within populations. No correlation was found between genetic diversity and aggressiveness or the geographic origin of the isolates. Results of the study indicate that pathogenic F. oxysporum are commonly associated with root rot of soybean with various aggressiveness and they are genetically diverse.


Introduction
Soybean [Glycine max (L.) Merr.] is an important oilseed crop and a valuable source of vegetable proteins (Loganathan et al., 2010).Approximately 7 million tons of soybean seeds were produced in 69 countries in the world (FAO, 2016).With the production of 693 thousand tons of soybean seeds, China is considered as one of the world's leading soybean producers (FAO, 2016).Total areas planted to soybean in China have been increasing, and the largest soybean producing province in the country is Heilongjiang (Li et al., 2013;Shurtleff & Aoyagi, 2016).Heilongjiang is located in northeast China with 4.3 million hectares of soybean produced in the province (Shurtleff & Aoyagi, 2016).
Soybean production is hampered by the occurrence of root rot disease caused by Fusarium spp.Fusarium root rot has been a problem in soybean production in many countries worldwide (Sinclair & Backmen, 1989;Arias et al., 2013;Zhang et al., 2013a).Tap and lateral roots infected by the disease often become reddish brown or light to dark brown.The roots become shallow and fibrous and eventually rotted, and plants may be wilted especially under conditions of low moisture and high temperature.Root rot of soybean can be induced by various Fusarium species, with F. oxysporum being the most common species reported (Nelson, 1999;Shiraishi et al., 2012;Zhang et al., 2013a).
Management of Fusarium root rot of soybean is difficult.Information regarding fungicides effective for suppressing F. oxysporum on soybean is limited.In northeast China, growers often abandon production of soybean when the fields get heavy infestation by the pathogen due to lack of effective disease control measures and panic of severe yield loss. Host resistance is a recommended strategy for managing soil borne diseases.In a study conducted in Canada (Zhang et al., 2013a), 70 soybean cultivars were evaluated under field conditions and 17 cultivars with the lowest severity of root rot caused by F. oxysporum, ranging from 1.3 to 2.2, were the most resistant.Soybean cultivars with resistance to Fusarium root rot under conditions in northeast China are not known to be available.It is unknown neither if F. oxysporum causing root rot of soybean are phenotypically and genetically diverse.It is highly desirable to determine diversity of the pathogen for developing effective disease management programs such as the use of host resistance.
Genomic fingerprinting techniques have been widely used to study genetic variability, population structure, and species phylogeny of F. oxysporum (Silva et al., 2013;Chen et al., 2014;Zimmermann et al., 2015).Among the techniques, amplified fragment length polymorphism (AFLP) is well recognized which has great discriminatory power (Vos et al., 1995;Mueller & Wolfenbarger, 1999;Silva et al., 2013).In a study on Fusarium wilt of bitter gourd caused by F. oxysporum f. sp.momordicae, AFLP analysis differentiated isolates with high virulence from those with low virulence, and pathogenicity of the 48 pathogen isolates was correlated with geographical locations of isolation of the isolates (Chen et al., 2014).In another study on F. oxysporum f. sp.radicis-cucumerinum from cucumbers, analysis with AFLP markers divided the 30 isolates in two distinct clusters (Tok & Kurt, 2010).All isolates in one cluster belonged to a vegetative compatibility group and isolates in the other cluster belonged to another vegetative compatibility group.These studies indicate that AFLP analysis is a useful tool in assessing genetic diversity of F. oxysporum populations.
The objective of this study was to determine pathogenicity of F. oxysporum associated with root rot of soybean in northeast China and use AFLP technology to analyze genetic diversity of the pathogen.This study advances our understanding of the etiology of the disease and population genetics of the pathogen, which provides valuable information for developing disease management programs.

Sample Collection and Pathogen Identification
Soybean plants showing root rot symptoms were sampled from different regions in Heilongjiang, China, in 2012 (Figure 1).Roots were washed with tap water, cut into pieces of 0.5 cm 3 , disinfested with 70% ethanol for 2 s and 0.5% NaOCl for 5 min, and rinsed three times with sterile distilled water.Root tissues were placed on potato dextrose agar (PDA) and incubated at 26 °C.Fungal hyphae grown from the tissues were transferred to fresh PDA plates.The isolates were identified as F. oxysporum according to cultural and morphological characteristics (Nelson et al., 1983;Leslie & Summerell, 2006).Single conidium isolates were obtained using the methods reported previously (Leslie & Summerell, 2006;Petkar et al., 2017).
For molecular identification, mycelia from 5-day-old cultures on PDA were used to extract DNA using the methods reported by Zhang et al. (2010) with minor modifications.In brief, mycelia of the isolates were pulverized using liquid nitrogen and homogenized using extraction buffer containing cetyl trimethyl ammonium bromide (CTAB), 0.5% charcoal along with 0.2% β-mercaptoethanol.After incubation at 65 °C for 15 min, homogenates were purified three times with chloroform:isoamyl alcohol (24:1).The upper aqueous phase (400 μL) was transferred to a tube containing 800 μL isopropanol, and DNA pellets were obtained by adding 0.67 volumes of propanol.The pellets were washed with ice-cold ethanol (70%), air dried, dissolved in 50 μL of deionized water, and stored at 4 °C.Polymerase chain reaction (PCR) analysis was performed with F. oxysporum species-specific primers FOF1 and FOR1 using the conditions described previously (Zhang et al., 2013b).PCR products were visualized by running 1.5% agarose gel and observed under a gel documentation system.
3.1 Modification of DNA and Template Preparation AFLP analysis was conducted using the methods of Vos et al. (1995) with minor modifications.To prepare DNA templates, 3 µL genomic DNA (150 ng/µL) was incubated for 4 h at 37 °C with 17 µL of restriction digestion mixture containing 3 U EcoRI (15 U/µL), 3 U MseI (10 U/µL), 2.0 µL 10 × PCR buffer with BSA, and 14.5 µL double-distilled water (ddH 2 O).Aliquots of 5 µL digested DNA were then analyzed by electrophoresis in a 1.2% agarose gel.In the digestion process, digestion products were ligated with EcoRI and MseI adapters at the following conditions: 95 °C for 5 min, 65 °C for 10 min, and 37 °C for 10 min.

Ligation Reaction
Before performing ligation reactions, restriction digestion mixture obtained from the previous step was incubated at 65 °C for 10 min to inactivate restriction enzymes.Each restricted DNA product was, then, incubated with 5 µL of ligation mixture, which consisted of 1.8 µL ATP, 0.5 µL EcoRI adapter mix, 1.0 µL MseI adapter mix, 1.0 µL T4 DNA ligase, 0.5 µL T4 DNA ligase buffer, and 0.2 µL ddH 2 O, at 37 °C for 12 h.A volume of 5 µL ligated products was loaded on a 1.2% agarose gel to check for complete ligation.DNA templates, which were completely digested and ligated, were used for AFLP reactions.
A total of 64 primer combinations were tested (8 EcoRI primers and 8 MseI primers) in this study and eight combinations, which generated clear bands with high polymorphism and good repeatability, were selected for genetic diversity analysis of the 50 F. oxysporum isolates.

Pathogenicity of Isolates
Fifty F. oxysporum isolates were collected from five different geographic areas of Heilongjiang province, China, including 10 from Harbin, 7 from Heihe, 5 from Jixi, 11 from Jiamusi, and 17 from Qiqihar.The majority of F. oxysporum isolates (68%) were obtained from soybean seedlings and the rest of the isolates were from mature soybeans (Table 2).When tested on soybean seedlings, significant variation in aggressiveness was observed among the isolates (Table 2).There were 15 weak pathogenic isolates (PDI < 25%), 21 medium aggressive isolates (25% ≤ PDI < 50%), and 14 highly aggressive isolates (PDI ≥ 50%).The most aggressive isolate was 103 (PDI = 82.9%)obtained from soybean seedling in Heihe.Isolates 12,93,33,147,163,167,125,90,91,166,150,137 and 144 had disease index lower than 10%.There was no correlation between aggressiveness of the isolates and their geographic origins, or between aggressiveness of the isolates and plant age (seedling or mature plant) when the fungus was isolated.

Discussion
Fusarium species are common soil borne pathogens that cause destructive diseases on a wide range of hosts (Nelson et al., 1997;El-Kazzaz et al., 2008).F. oxysporum is frequently isolated from soybean roots and serious yield impact of root rot associated with Fusarium spp. is reported in many soybean producing areas (Wrather et al., 2001;Li & Ma, 2011;Arias et al., 2013).In the present work, F. oxysporum was isolated from all the sampling sites in Heilongjiang province, suggesting that this pathogen is one of the most important causal agents of soybean root rot in this region.Our findings are in line with previous studies which showed F. oxysporum to be the predominant species recovered from diseased soybean plants in China (Bai et al., 2009;Li & Ma, 2011) and other countries (Leslie et al., 1990;Nelson, 1999;Zhang et al., 2013a).
Variations in aggressiveness among pathogenic strains of F. oxysporum from soybean root were reported in a few studies.In a study to assess impact of Fusarium species on soybean yield and growth, significant difference was observed among the F. oxysporum strains in their ability to cause root rot and damping-off of soybean (Arias et al., 2013).Ellis et al. (2016) reported that 3 isolates of F. oxysporum caused high incidence of vascular discoloration of soybean stems or roots and 25 strains caused low to moderate levels of incidence, and one of the fungal effector genes, Six6, was found only in the strains causing high levels of vascular wilt.Of the 50 strains obtained from Heilongjiang province, 14 strains caused severe root rot, 21 strains were moderately aggressive and 15 strains induced low root rot severity on soybean, indicating significant variations in aggressiveness among the strains.
Studies to assess genetic diversity of F. oxysporum from soybean root are limited.In studies on F. oxysporum from other crops analyzed by AFLP, genetic groups were found to be associated with aggressiveness of F. oxysporum f. sp.momordicae isolates (Chen et al., 2014) or vegetative compatibility group of F. oxysporum f. sp.radicis-cucumerinum isolates (Tok & Kurt, 2010).AFLP was used in this study to access genetic diversity of F. oxysporum isolates in major soybean producing regions in Heilongjiang.The eight primer pairs used resulted in 99% polymorphic fragments, which is in agreement with previous reports that AFLP generates more polymorphic bands than other DNA-fingerprinting techniques (Vaneechoutte, 1996;Duan et al., 2008).AFLP analysis demonstrated great variability in genetic relatedness among the 50 F. oxysporum strains from soybean, with 7 genetic groups identified at a similarity level of 0.2.AMOVA analysis indicated that most of molecular variation was attributed to within populations, suggesting high level of genetic variation and complexity of the F. oxysporum isolates.AFLP groups were not correlated with geographic location of isolation, plant age of isolation, or aggressiveness of the isolates, as suggested by low F ST values (< 0.03).
In a study of Fusarium oxysporum f. sp.lentis isolates from lentil plants, the isolates were clustered in two AFLP groups, and no correlation was found between AFLP grouping and geographical origin or aggressiveness of the isolates (Belabid et al., 2004).AFLP analysis was conducted to study mutation of F. oxysporum f. sp.vasinfectum isolates associated with cotton, and there was no correlation between the AFLP mutations and increased aggressiveness of the isolates (Wang et al., 2008).Duan et al. (2008) reported that AFLP groups of F. oxysporum f. sp.niveum isolates were not associated with geographic regions of the isolates but were correlated with their physiological races.These studies indicate that AFLP grouping may or may not be related to phenotypic traits of F. oxysporum isolates, depending on the pathogens and host plants studied.

Conclusion
Soybean root rot is a serious disease causing significant yield reduction in soybean production.Results in the study indicate that there is a high level of genetic diversity within F. oxysporum strains responsible for soybean root rot in Heilongjiang province, China.The strains differ in aggressiveness that is not associated with their geographic distribution, plant age of isolation, and molecular fingerprinting groups.The information generated in the study advances our knowledge about etiology, pathogenicity, and diversity of soybean root rot pathogen, which facilitates development of effective strategies for managing this major disease.

Table 1 .
Sequences of adapters and primers used for AFLP analysis Agricultural Sci f Fusarium oxy ng 125 g of so days.A mycel The flasks we zed by F. oxysp ience ysporum in Hei orghum seeds a lial plug (7 mm ere incubated a porum were un h a 0.5 cm lay s were used.Tw

Table 2 .
Pathogenicity of Fusarium oxysporum isolates on soybean under greenhouse conditions

Table 3 .
Hierarchical partitioning of variance among and within population groups of Fusarium oxysporum strains based on analysis of molecular variance (AMOVA)