A study on the pathogen species and physiological races of tomato Fusarium wilt in Shanxi, China

CHANG Yin-dong, DU Bin, , WANG Ling, JI Pei, XIE Yu-jie, LI Xin-feng, LI Zhi-gang, , WANG Jianming

1 College of Agriculture, Shanxi Agricultural University, Taigu 030801, P.R.China

2 Department of Horticulture, Taiyuan University, Taiyuan 030051, P.R.China

3 Technology Center Yuncheng Sub-Center, Shanxi Entry-Exit Inspection and Quarantine Bureau, Yuncheng 044000, P.R.China

4 Department of Genetics and Biochemistry, Clemson University, Clemson 29634-0318, South Carolina, USA

1. Introduction

As one of the most popular fruit vegetables in the world,tomato is the main crop planted in the vegetable producing areas in the north of the Yangtze River, China. In recent years, with the rapid development of fresh tomato and its products consumption, the continuous cropping problem and soil borne diseases of tomato are becoming more serious. Tomato Fusarium wilt has become a limiting factor to the sustainable development of tomato production due to the long-term survival of the chlamydospore in the plant debris of the soil. This could infect tomato plants during its growth period and pose a serious impact on its production(Armstronget al.1978; Chen 1982; Xunet al.2008).

Both domestic and foreign studies have shown that tomato Fusarium wilt is mainly caused byF. oxysporumf.sp.lycopersiciand has obviously physiological differentiation(Armstronget al.1978; Liuet al.2013). In addition,F. solani,F. verticillioidesandF. proliferatumcan also infect tomatoes causing plant withering (Liet al.2011; Zhanget al.2016).Currently, there are 3 physiological races ofF. oxysporumf. sp.lycopersicithat have been reported. The main pathogen of tomato Fusarium wilt in China isF. oxysporumf. sp.lycopersicirace 1 (Liuet al.1991; Wanget al.1996b;Zheng and Wang2000), and race 2 was only found in Zhejiang and Taiwan of China (Xuet al.2000; Sheu and Wanget al.2007). Through comparing the root colonization processes of pathogenic and non-pathogenic strains ofF. oxysporumin tomato seedlings, it was revealed that the seedlings possess different resistance to the 2 strains(Olivain and Alabouvette 1999). Maet al.(2010), through the whole genome sequence comparison, found that there are 4 lineage-specific chromosomes in the whole genome ofF. oxysporumand these chromosomes showed huge difference based on different host-specificities. To unveil the base of pathogenicity difference between the physiological race 1 and race 4 ofF. oxysporumf. sp.cubense, different patterns on gene content and gene expression have been shown by contrasting the genome and transcriptome (Guoet al.2014). Therefore, accurate identification of the tomato Fusarium wilt pathogens and clarification of its physiological differentiation has an important theoretical and practical value to the pathogen identification and disease resistance breeding.

Shanxi Province is located in Loess Plateau of China with varied topographies and changeable climate which contributes to the difference of ecological environments.Since tomato planting area in Shanxi is wide, the occurrence of Fusarium wilt is high, but its pathogen species and physiological races are not yet detailedly studied. Traditionally, pathogenicity test is usually applied to identify the formae speciales and physiological races ofF. oxysporumf. sp.lycopersici, which is time-consuming and susceptible to environmental conditions. With continuous efforts of researchers,EF-1αsequence analysis has been proved as an efficient measure to identifyFusariumisolates at species level (Geiseret al.2004; O’Donnellet al.2010; Zhaoet al.2011; Huet al.2013; Wanget al.2017). At present, the rapid development of molecular biology technology enables researchers to develop specific molecular markers for rapid detection and identification of different formae speciales and physiological races ofF. oxysporum(Lievenset al.2008; Baysalet al.2010;Linet al.2013; Zhanget al.2013; Ayukawaet al.2016).Therefore, exploring the use of molecular technology for the identification of formae speciales or physiological races has a great scientific significance for rapid and accurate detection of physiological differentiation of pathogens.

In this study, the main pathogens of tomato Fusarium wilt in different areas of Shanxi Province and its physiological differentiation was studied by morphological and molecular identification methods.

2. Materials and methods

2.1. Test isolates and tested primers

A total of 105Fusariumisolates were recovered from Fusarium wilted tomato plants collected in 17 districts of Shanxi Province, China. The number of isolates and geographic origins are shown in Table 1.

Root systems of infected tomato plants were collected in Shanxi between June 2014 and September 2017 (Table 1).

Primers used for discrimination ofFusariumspecies,F. oxysporumand physiological races ofF. oxysporumf.sp.lycopersiciare shown in Table 2.

2.2. Isolation of pathogens from infected tissues

Tissue culture method and moisturizing culture method were used to isolate the infected root systems and single-spore isolates were obtained using the procedure described by Fang (1998).

2.3. Morphological identification

The purified isolates were inoculated on potato sucrose agar(PSA) and cultured at 25°C for 4 days with 3 replicates. The colony diameter, aerial mycelium properties, and colony color were recorded at days 5 and 7. Meanwhile, the existence,amounts, size and shape of microconidia, macroconidia and chlamydospores were characterized under a microscope.Then, the isolates were classified by referring to the relevant taxonomic literatures (Booth 1971; Wanget al.1996a; Leslie and Summerell 2006; Lvet al.2010).

2.4. Molecular biology identification

Species identification based on EF-1α sequence analysisThe genomic DNA was extracted by modified cethyl-trimethyl ammonium bromide (CTAB) procedure(Wang 2016), dissolved in 100 μL TE buffer, and stored at–20°C.

Based on the results of morphological identification,18 representative strains were chosen to carry out PCR amplification and sequencing ofEF-1αbased on the previously described measures (Wanget al.2017). AllEF-1αsequences obtained in the study were used to initially identify these isolates to the species level in GenBank Database and Fusarium-ID Database, respectively. Taken theEF-1αsequence ofNeonectria ramulariaeas the outgroup, the high homology and reliable sequences were downloaded to construct neighbor-joining tree as described previously (Wanget al.2017). The chosen isolates were further verified by means of phylogeny analysis.

Table 1 Tested isolates and collection sites in Shanxi Province, China

Table 2 Primers used in the test

ldentification of Fusarium oxysporumThe specific band ofF. oxysporumwas amplified using the general primer pair FOF1/FOR1. The PCR amplifications were performed in a 20-μL mixture containing 10 μL of 2×TaqPCR Master Mix,1 μL of each primer (10 μmol L–1), 1 μL of DNA template,and 7.0 μL of ddH2O. The reactions were performed with a thermal cycler (MyclerTM, Bio-Rad, Hercules, California,USA) using the thermal program as described previously(Mishraet al.2003). PCR products were detected by 2%agarose gel electrophoresis.

ldentification of physiological races of F. oxysporum f. sp. lycopersiciThe specific bands for physiological races ofF. oxysporumf. sp.lycopersiciwere amplified using 2 specific primer pairs sp13f/r and sp23f/r. The PCR amplification was performed in a 25-μL mixture containing 2.5 μL of 10×Taqbuffer, 1 μL of each primer (10 μmol L–1),2 μL of dNTP, 0.3 μL ofTaqpolymerase, 1 μL of DNA template,and 17.2 μL of ddH2O. The reactions were performed in the above mentioned equipment using the reaction program as described previously (Hirano and Arie 2006). PCR products were detected by 2% agarose gel electrophoresis.

2.5. Pathogenicity testing

Ten representative isolates recovered from tomato diseased plants and one tomato susceptible cultivar were selected for pathogenicity testing. A total of 20-day-old healthy seedlings were inoculated by pouring the wound root with spore suspension. To slightly injure the root, a sterilized scalpel was inserted to 3 cm depth on both sides of the seedlings root. Then, the injured root was inoculated with 106conidial mL–1of spore suspension. Instead, sterile water was used to control seedlings. Each selected isolate was inoculated with at least 3 replicates. Plants were maintained in the artificial climate chamber where dark and light cycles were 14 h/10 h at 28–25°C for 14 days. Timely watering was done to keep the substrate moist. The disease index was calculated as per the disease grading standards of tomato Fusarium wilt symptom at seedling stage (Table 3) (Xieet al.2002) and the pathogens were re-isolated and identified using Koch’s postulates to determine its pathogenicity.

3. Results

3.1. Morphological identification results

Morphological identification results are shown in Table 4 and Fig. 1. As shown in Table 4, 7 species ofFusariumwere identified within 105 isolates, namely,F. oxysporum(56 isolates),F. solani(20 isolates),F.verticillioides(13 isolates),F. subglutinans(8 isolates),F. chlamydosporum(4 isolates),F. sporotrichioides(3 isolates) andF. semitectum(1 isolate). As shown in Fig. 1, the isolation frequencies of differentFusariumspecies were significantly different among whichF. oxysporumhad the highest isolation frequency of 53.3%. The isolation frequencies of other species were decreasing:F. solani19.0%,F.verticillioides12.4%,F. subglutinans7.6%,F. chlamydosporum3.8%,F. sporotrichioides2.9%, andF. semitectum1.0%.

As shown in Table 4 and Fig. 1, the species differences of theFusariumpathogens of tomato in different districts of Shanxi can be noticed. For each district, at least 2Fusariumspecies could be isolated. In Jinzhong and Taiyuan, except of common species such asF. oxysporum,F. solani,F.verticillioides, andF. sporotrichioides,F. subglutinansandF. semitectumwere isolated from Jinzhong, whereasF. chlamydosporumwas isolated from Taiyuan. ThreeFusariumspecies were isolated from Lüliang, Yuncheng,Datong and Linfen, among whichFusariumspecies were the same in Lüliang, Yuncheng and Datong, includingF. oxysporum,F. solaniandF.verticillioides.Instead ofF.verticillioides,F. chlamydosporumwas isolated from Linfen. TwoFusariumspecies were isolated from the rest of the districts among whichF. oxysporumandF. verticillioidesisolated from Yangquan and Changzhi. In addition, except for common speciesF. oxysporum,F. subglutinansandF. solaniwere isolated from Xinzhou and Shuozhou,respectively (Fig. 1).

3.2. Molecular biology identification results

ldentification results of Fusarium speciesBased on the homologous blast and phylogeny analysis, the results showed that the homologous level of the 18 isolates chosen and homologous isolates reached 92–99% (Table 5), all of the chosen isolates were clustered with homologous isolates (Fig. 2). The tested isolates were identified to 7Fusariumspecies, which further verified the morphological identification results (Table 5).

ldentification results of F. oxysporumAs shown in Fig. 3,isolates yielded the expected single band (290–320 bp)in the PCR assay using specific primers FOF1/FOR1 recognized forF. oxysporum. A total of 56 isolates from all of the isolates were identified asF. oxysporum(Table 6), as with morphological identification results.

Table 3 Grading standards of tomato Fusarium wilt symptom at seedling stage

Table 4 Morphological identification results

ldentification results of physiological races of F. oxysporum f. sp. lycopersiciAs shown in Fig. 4,isolates only yielding the expected single band (445 bp)with specific primers sp13f/r were recognized as race 1,isolates only yielding the expected single band (518 bp) with specific primers sp23f/r were recognized as race 2, whereas the isolates yielded the expected bands with both primer pairs sp13f/r and sp23f/r respectively were recognized as race 3. The isolate without band was initially recognized as non-F. oxysporumf. sp.lycopersici. A total of 29, 5, and 6 isolates were identified as race 1, race 2, and race 3 with frequencies of 51.8, 8.9, and 10.7%, respectively. The results showed that there were 3 physiological races in Shanxi Province among which race 1 was dominant.

As shown in Fig. 5, there were significant differences in the species and amounts of the physiological races ofF. oxysporumf. sp.lycopersiciin different districts of Shanxi.All of 3 physiological races ofF. oxysporumf. sp.lycopersicicould be detected from isolates collected in Jinzhong,Taiyuan and Yuncheng. Except for Changzhi, race 1 could be detected from all the other chosen districts. Except for Datong and Shuozhou, isolation frequencies for race 1 in other districts were significantly higher than other races,which was more than 50%. Race 2 could be detected in Jinzhong, Taiyuan, Yuncheng and Datong with the isolation frequency in Datong being 66.6% which was significantly higher than that of other races. Race 3 was detected only in Jinzhong, Taiyuan and Yuncheng among which the maximum isolation frequency was 33.3% corresponded to Taiyuan.

Fig. 1 Species and amounts of isolates of Fusarium recovered from wilted tomato plants in 10 cities of Shanxi, China.

Table 5 Molecular discrimination results of Fusarium species

3.3. Pathogenicity testing results

The disease index and incidence of tomato seedlings after 15 days of inoculation are shown in Table 7 and Fig. 6.Pathogenicity ofF. oxysporumf. sp.lycopersiciwas much higher than the other species and pathogenicity of race 2 for the same specie was the highest. The disease indexes of tomato seedling treated by the 3 races were 57.1, 64.3 and 46.0, respectively. The disease index of the otherF. oxysporumwas only 26.5 which revealed a relatively weak pathogenicity. The pathogenicity ofF. solaniandF. verticillioideson tomato seedlings was also strong and the disease indexes of the seedlings treated by 2 species were 46.5 and 44.8 respectively. The pathogenicity ofF. subglutinanson tomato seedling was moderate, and the disease index was 38.0. Tomato seedlings inoculated withF. chlamydosporum,F. sporotrichioides, andF. semitectumshowed relatively mild symptoms and the disease indexes were 17.3, 15.0 and 14.0, respectively. For the non-inoculated control (CK), a few plants appeared with symptoms grade 1 or 2 and their disease index was 6.1(Tables 3 and 7).

Fig. 2 Neighbor-joining (NJ) phylogenetic tree of Fusarium species from Shanxi, China inferred from EF-1α sequences. The numbers at the nodes represent bootstrap values estimated from 1 000 replications (greater than 50%). Fusarium incarnatum is a synonym of Fusarium semitectum. Neonectria ramulariae (HM054091.1) was used as an outgroup. The scale bar indicates 2% sequence divergence.

Fig. 3 Specific amplification products using specific primers FOF1/FOR1 to detect Fusarium oxysporum isolates.

As verified by the Koch’s postulates with the exception of the CK treatment, all of the tested isolates were isolated from the root of inoculated tomato seedlings. In this test,F. oxysporumf. sp.lycopersici,F. solani,F. verticillioides,F. subglutinans, and otherF. oxysporuminfected the tomato seedlings at 14 days after inoculation, and induced obvious wilt symptoms. The disease index was significantly different from that of CK treatment, which was identified as the pathogens of tomato Fusarium wilt.The disease indexes of tomato seedlings inoculated withF. chlamydosporum,F. sporotrichioides, andF. semitectumwere low with no significant wilt symptoms, and there was no difference between those treatments and there was no significant difference between those treatments and the CK treatment (Table 7). Therefore, these 3 species were not recognized as the pathogen of tomato Fusarium wilt.

Table 6 Molecular discrimination results of Fusarium oxysporum (F. o.)

Fig. 4 Specific amplification products using specific primer sets sp13f/r and sp23f/r to detect physiological races of Fusarium oxysporum f. sp. lycopersici.

4. Discussion

In recent years, researchers have found that except forF. oxysporumf. sp.lycopersici, otherFusariumspecies can also infect tomato resulting in complex infections(Liet al.2011). Zhanget al.(2016) have found that in addition to the dominant species ofF. oxysporum,F. solaniandF. graminearumcan also infect tomato and that the induced symptoms are very similar, but the virulence is relatively weaker thanF. oxysporum, which is consistent with the results of this study. In this study, the frequency ofF. chlamydosporum,F. sporotrichioidesandF. semitectumwere relatively low, and the disease index of tomato seedlings inoculated with these 3 species were not significantly different from that of the control group.Therefore, the pathogenicity of these species to tomato needs further study.

Fig. 5 Distribution and amounts Fusarium oxysporum and 3 races of F. oxysporum f. sp. lycopersici in 10 cities of Shanxi,China.

For a long time, the classification and identification ofFusariumin China was mainly based on the relevant systems ofFusariumclassification, such asThe Genus Fusarium(Booth 1971),Guide to the Identification of Common Fusarium(Wanget al. 1996a) andThe Fusarium LaboratoryManual(Leslie and Summerell 2006). The identification of formae speciales and physiological races ofF. oxysporumare mainly based on pathogenicity differentiation. This method is time-consuming and vulnerable to environmental conditions. The use of molecular means can overcome the problems mentioned above, which is an important means to identify the physiological race. A large number of studies have shown that the nucleotide sequences of the internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS) are not suitable for distinguishing between different formae speciales and physiological races (Wanget al.2013), and just a few gene loci can be used to effectively distinguish the physiological races by sequence analysis (Jacobset al.2013; Zenget al.2014). Nowadays, DNA molecular marker, toxic genes,and transposon-specific primers are increasingly used in formae speciales and physiological races ofF. oxysporum(Lievens 2008; Liet al.2012; Zhanget al.2013; Ayukawaet al.2016). Mishraet al.(2003) developed a specific primer ofF. oxysporum, which is now widely used to specifically identifyF. oxysporumfrom otherFusariumspecies. Hirano and Arie (2006) developed specific primers sp13f/r and sp23f/r by comparing the partial nucleotide sequences of the endo-polygalacturonase of different physiological races to differentiate three races. With these primer pairs, Baysalet al.(2010) investigated the races ofF. oxysporumf. sp.lycopersiciin the western Mediterranean region of Turkey,and identified the main pathogens of tomato Fusarium wilt in the area to be race 2 and race 3. Furthermore,these primer pairs were also used in the identification of physiological races ofF. oxysporumf. sp.lycopersiciin Northeast Brazil and Chile (Barbozaet al.2013;Sepúlvedachaveraet al.2014). This experiment, through the molecular identification method, showed that in addition to race 1 and race 2, there is also race 3 in Shanxi, which is the first report of physiological race 3 causing tomato Fusarium wilt in China. These races also need to be further verified by the differentiation of pathogenicity to differential hosts and the overall distribution of physiological races in Shanxi has to be studied in detail in the future.

Table 7 Disease index of tomato seedling 15 days after inoculation of different isolates

Fig. 6 Growth conditions of tomato seedling 15 days after inoculation of different isolates. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and CK denote the tomato seedlings inoculated with Fusarium oxysporum f. sp. lycopersici race 1, race 2, and race 3, F. oxysporum,F. solani, F. verticillioides, F. subglutinans, F. chlamydosporum, F. sporotrichioides, F. semitectum, and blank control, respectively.

Numerous studies have shown that the physiological differentiation ofF. oxysporumis very obvious (Armstronget al.1978; Liuet al.2013; Wanget al.2013). For the 56 isolates ofF. oxysporum, 16 isolates were not identified as physiological races ofF. oxysporumf. sp.lycopersici,which could be another formae speciales or kind of new physiological races, and/or could be a limitation of the versatility of the selected primers. The reasons for aspects mentioned above are yet to be further explored.

5. Conclusion

The study showed that there were 4 pathogens causing tomato disease in Shanxi, which wereF. oxysporum,F. solani,F. verticillioidesandF. subglutinans.Among them,the isolation frequency ofF. oxysporum, accounting for 53.3%, was significantly higher than other species. There were 3 physiological races ofF. oxysporumf. sp.lycopersiciin Shanxi, with race 1 being the dominant race. The distributions ofF. solani,F. verticillioides, andF. subglutinanswere relatively broad, the frequency of those species was relatively high, accounting for 39% of the total isolates, and the pathogenicity of those species to tomato seedlings was relatively high, being also important pathogens of tomato.

Acknowledgements

This research was partially supported by the Shanxi Provincial Science and Technology Planning Project,China (20120311019-3), the Shanxi Provincial Science and Technology Foundation Platform Construction Project, China(1105-0104), and the Shanxi Provincial Graduate Education Innovation Project, China (2017BY065).

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