DENG Dong,SUN Su-li,DU Chen-zhang,XIANG Chao,LONG Jue-chen,CHEN Wei-dong,ZHU Zhen-dong

1 Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,P.R.China

2 Chongqing Academy of Agricultural Sciences,Chongqing 402160,P.R.China

3 Crop Research Institute,Sichuan Academy of Agricultural Sciences,Chengdu 610066,P.R.China

4 United States Department of Agriculture-Agricultural Research Service,Washington State University,Pullman,WA 99164,USA

Abstract White mold of pea caused by Sclerotinia sclerotiorum is a common disease in China.However,we discovered that the diverse Sclerotinia species could cause white mold on pea plants in Chongqing and Sichuan of China during recent disease surveys.Thus,the objective of this study was to confirm the causal agents from diseased pea plants.The obtained isolates of white mold from Chongqing and Sichuan were identified by morphological characters and molecular characterization to determine the pathogen species,and their pathogenicity was confirmed on pea through completing Koch’s postulates.Fungal isolates of Sclerotinia-like were obtained from diseased plants or sclerotia.Based on morphological characteristics and molecular characterization,30 isolates were identified to three species,six isolates as S.minor,seven as S.sclerotiorum,and 17 as S.trifoliorum.In pathogenicity tests on pea cultivars Zhongwan 4 and Longwan 1,all 30 isolates caused typical symptoms of white mold on the inoculated plants,and the inoculated pathogens were re-isolated from the diseased plants.This study confirmed that white mold of pea was caused by three Sclerotinia species,S.sclerotiorum,S.minor and S.trifoliorum in Chongqing and Sichuan. It is the first report that S.minor and S.trifoliorum cause white mold of pea in Southwest China.

Keywords:Pisum sativum,white mold, Sclerotinia sclerotiorum,Sclerotinia minor,Sclerotinia trifoliorum

1.Introduction

Pea (Pisum sativmL.) is one of the most important legume crops,and is widely planted in the world.China is the main pea production country with more than 50% fresh pea producing area of the world (FAOSTAT 2019).However,the problem of diseases has become increasingly prominent with the increase of pea planting areas.White mold,caused by theSclerotiniaspp.,is a major disease of pea in Chongqing and Sichuan.The disease,thriving in a cool and moist environment,occurs from seedling to adult stage,and is most serious during late flowering growth stage (Kraft and Pfleger 2001).

Sclerotiniaspp.,with a global distribution,can cause white mold,stem rot or crown rot on many crops and wild plant species.There are three important species ofSclerotinia,S.sclerotiorum(Lib.) de Bary,S.minorJagger andS.trifoliorumErikss.,causing significant economic losses in agricultural and horticultural production.Sclerotinia sclerotiorumis the most economically harmful among the three species,infecting more than 600 plant species including many economically important crops (Boland and Hall 1994;Liang and Rollins 2018).Sclerotinia minorcauses diseases in at least 94 plant species (Melzeret al.1997).Compared with the other two species,S.trifoliorumhas a more restricted host range,mainly infecting legumes (Kohn 1979;Prattet al.1988).The three species all were reported to cause white mold of pea in India,Brazil,Canada,and the United States,but the most commonly reported species isS.sclerotiorum(Kohn 1979;Prattet al.1988;Boland and Hall 1994;Melzeret al.1997).In China,onlyS.sclerotiorumis reported to cause white mold on pea to date (Tai 1979;Wu 1988).

Identification ofSclerotiniaspp.in history has mainly been based on the morphological characteristics of sclerotia and ascospores (Kohn 1979).Sclerotinia minorproduces small sclerotia,which make it easy to distinguish it from the other two species (Chenaultet al.2009).Sclerotinia sclerotiorumandS.trifoliorumcan be distinguished by ascospore morphology (Kohn 1979;Prattet al.1988).However,production of ascospore ofSclerotiniaspp.in the laboratory was very tedious and time-consuming,and it might take up several months(Kohn 1979;Njambereet al.2008;Baturo-Ciesniewskaet al.2013).In addition,some isolates ofS.trifoliorumwere heterothallic and required a compatible mating strain for inducing carpogenic germination (Vleugelset al.2012).

In order to increase the efficiency ofSclerotiniaspp.identification,several different techniques have been used for rapid and accurate identification ofSclerotiniaspecies,mainly based on the molecular methods (Baturo-Ciesniewskaet al.2017).Tariqet al.(1985) reported a difference in the growth rate betweenS.sclerotiorumandS.trifoliorum.Sclerotinia trifoliorumgrows slower thanS.sclerotiorumat 25°C.Powerset al.(2001) found thatS.trifoliorumcontains several introns near the 5´ end of the rDNA nuclear small-subunit that is included in the amplification of the internal transcribed spacer (rDNAITS) region.Thus,the PCR products of the amplified rDNA-ITS sequences could distinguishS.trifoliorumfromS.sclerotiorumandS.minor.Based on sequence variation of laccase 2 (Lcc2),aspartyl protease (Aspr)and calmodulin (Cad) genes,Abd-Elmagidet al.(2013)developed the specific primers to rapidly identifySclerotiniaspecies.

In China,S.sclerotiorumhas been recorded on pea in Guangxi,Guizhou,Sichuan,Zhejiang,Jiangxi and Hubei provinces/autonomous region and Taiwan of China(Tai 1979;Wu 1988;Liet al.1996).Sclerotinia minorandS.trifoliorumwere only documented on pea in Hubei Province,but the pathogens from pea have not been identified in detail yet (Liet al.1996;Yanget al.2016).The objective of the present study was to identify pathogen species inciting white mold of pea in Chongqing and Sichuan by using morphology and molecular characterization.

2.Materials and methods

2.1.Disease survey and sample collection

In March 2019,field disease surveys of pea at adult stage were conducted in Chongqing and Sichuan of China.We found that some diseased pea plants showSclerotinia-like symptoms spotted in some fields located in Yongchuan(29.35´N,105°92´E) and Hechuan Districts (29°97´N,106°27´E) in Chongqing and Yilong County (31.27´N,106°29´E) in Sichuan.To confirm the causal agents,the diseased plant tissues and sclerotia were collected from these fields,which were wrapped in filter paper and placed in ziplock bags for later pathogen isolation.

2.2.Pathogen isolation

These diseased plant tissues or sclerotia were sterilized in 75% ethanol for 30 s,followed by 2% sodium hypochlorite bleach for 2 min,rinsed three times in sterilized distilled water,dried on sterilized filter paper,and incubated (in 90-mm Petri dishes) on potato dextrose agar (PDA)containing 0.1% lactic acid at 22°C in the dark.After 2-3 d,fungi growing from the diseased tissues or sclerotia that were purified by transferring hyphal tips to new PDA dishes,and incubated under the same conditions.Sclerotia were collected from PDA dishes after 30 d,airdried and stored in centrifuge tube at -20°C for future use.

2.3.Growth rates and characteristics of sclerotia

The 7-mm-diameter mycelial plugs of each isolate were taken from the edge of a 2-d-old actively growing colony by a puncher.The plugs were transferred to the center of a 90-mm-diameter PDA dish and were cultured at 22°C in the dark.Colony diameters were measured every 12 h by averaging from two measurements taken at right angles to each other until colonies covered the whole dishes(Njambereet al.2008).This test was done in a random design with three replicates (three dishes per isolate)and repeated twice.The characteristics of colonies was recorded at 36 h,and the size of sclerotia was measured at 18 d.

2.4.Molecular identification

Total genomic DNA of all isolates was extracted from mycelium using the Fungi Genomic DNA Extraction Kit(Solarbio,Beijing,China) following the manufacturer’s instructions.The ITS region was amplified using primer pair ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) and ITS5(5´-GGAAGTAAAAGTCGTAACAAGG-3´) (Njambereet al.2008).PCR was performed using 3.2 µL of dNTP mix (2.5 mmol L-1),0.4 µL ofTaqpolymerase (5 U µL-1),2 µL of genomic DNA (10 ng µL-1),4 µL of polymerase buffers (10 U µL-1,TaKaRa,Japan),2 µL of each primer(25 µmol L-1).The total volume was adjusted to 40 µL with ddH2O and the PCR was conducted in a GeneAmp PCR System 9700 (PE Applied Biosystem,USA).The PCR amplification program includes the following steps:94°C for 5 min,35 cycles of 94°C for 1 min,55°C for 1 min,72°C for 1 min and a final extension at 72°C for 10 min.In addition,three pairs of specific primers are used to distinguishSclerotiniaspecies.Primer pairs SMLcc2F (5´-CCCTCCTATCTCTCTTCCAAACA-3´) and SMLcc2R (5´-TGACCAATACCAATGAGGAGAG-3´) forS.minor;SSasprF (5´-CATTGGAAGTCTCGTCGTCA-3´)and SSasprR (5´-TCAAACGCCAAAGCTGTATG-3´)forS.sclerotiorum;STCadF (5´-TCCTAGATCGACTCT CCTCCTTT-3´) and STCadR (5´-TCAAACGCCAAAGCT GTATG-3´) forS.trifoliorumwere used for amplifications of theLcc2,AsprandCadgene regions,respectively(Abd-Elmagidet al.2013).The PCR reaction system and program were the same as rDNA-ITS amplification except that the annealing temperature was 60°C.The sizes of PCR products were assessed by 1.5% agarose gel electrophoresis along with standard DNA markers.Gel was added with the Gelgreen Nucleic Acid Gel Stain (Biotium,USA) during production so that it can be observed and photographed directly under UV light.The rDNA-ITS amplified products of 10 representative isolates JH1,JH3,JH7,JH8,JH9,JH13,JH16,JH23,JH26,and JH29 were purified and sent to Sangon Biotech (Shanghai)Co.,Ltd.for sequencing.The resulting sequences were blasted in NCBI Database (http://www.ncbi.nlm.nih.gov).Phylogenetic analysis based on rDNA-ITS sequences using the MEGA X with UPGMA method and Tamura-Nei distance model (Mandal and Dubey 2012).

2.5.Pathogenicity test

Pathogenicity of isolates was determined on pea cultivars Zhongwan 4 and Longwan 1.Five seeds were planted in each paper cup (500 mL) filled with fresh vermiculite and the planted cups were placed in the greenhouse for 14 d at 21-25°C.Each isolate was cultured on PDA dishes in dark at 22°C for 2 d.The 5-mm-diameter mycelia plugs were then cut from the edge of actively growing colony of every isolate.These agar plugs were placed onto the 4th node of all 14-d-old pea plants in each cup at the attachment point where the leaf branches from the main stem (Porteret al.2009).

After inoculation,the plants were placed in a mist room with 100% relative humidity and temperature around 22°C for 3 d and the lesion length was then measured.The plants were transferred to a greenhouse with temperatures at 15-25°C,held for another 14 d,and then their survival were calculated (Porteret al.2009).All pathogenicity tests were repeated twice.

3.Results

3.1.Disease symptoms

During the field surveys in Chongqing and Sichuan,we found that theSclerotinia-like disease was severe and prevalent on pea,with the incidence nearly 40% in some fields (Fig.1-A).The leaves,stems and pods of diseased pea plants were chlorosis and became grayish green,tan,white and necrotic,and some parts were covered by white mold (Fig.1-B,C and D).The black sclerotia were irregular in shape and produced on the diseased tissue,usually on the necrotic stems and pods (Fig.1-B and D).

Fig.1 Symptoms of white mold on pea caused by Sclerotinia species in the field.A,pea plants infected by Sclerotinia showing wilt and death in the field.B,the leaves and stems became grayish green,necrotic and were covered by white mold.C,the lesions on leaves and stems become tan or white and the sclerotia appeared.D,white mold and sclerotia produced on the necrotic pods.

3.2.Cultural and morphological characteristics

ThirtySclerotinia-like isolates were obtained from Yongchuang and Hechuan of Chongqing and Yilong of Sichuan.On PDA medium,the isolates could be divided into three different groups according to growth rate and sclerotia size (Table 1).Fast-growing group including seven isolates,JH9,JH10,JH11,JH12,JH13,JH14,and JH15,had an average growth rate of (50.4±1.8) mm d-1and covered the entire surface of the PDA plates within 48 h (Fig.2-A).Moderate-growing group contained JH1,JH2,JH3,JH4,JH5,and JH6,with an average growth rate of (41.5±1.8) mm d-1and covered the agar surface in nearly 60 h (Fig.2-B).Slow-growing group comprised JH7,JH8,JH16,JH17,JH18,JH19,JH20,JH21,JH22,JH23,JH24,JH25,JH26,JH27,JH28,JH29,and JH30.The 17 isolates were with an average growth rate of(31.9±2.0) mm d-1and took about 72 h to cover the dishes(Fig.2-C).The average sclerotia size of the slow-growing group was (3.6-8.1) mm×(2.9-6.0) mm,which was the largest of the three groups (Fig.2-F).The fast-growing isolates had sclerotia with (3.1-5.8) mm×(2.4-4.7) mm in size (Fig.2-D),whereas moderate-growing isolates had sclerotia with (0.5-0.9) mm×(0.3-0.8) mm in size,which was significantly smaller than the other two groups(Fig.2-E).Previous studies showed thatS.sclerotiorumhad the largest colony and fast growth rate,followed byS.minorandS.trifoliorum(Tariqet al.1985),the sclerotial size ofS.sclerotiorumwas slightly smaller than that ofS.trifoliorum,andS.minorhad the smallest sclerotia(Prattet al.1988).Based on growth rate and sclerotia size,JH1 to JH6 could beS.minor;JH7,JH8,and JH16 to JH30 might beS.trifoliorum;and JH9 to JH15 should beS.sclerotiorum.

Fig.2 Morphological characterization of three Sclerotinia species.A,colony characteristics of S.sclerotiorum.B,colony characteristics of S.minor.C,colony characteristics of S.trifoliorum.D,sclerotia of S.sclerotiorum.E,sclerotia of S.minor.F,sclerotia of S.trifoliorum.Bars:1 cm.Photos of the colonies were taken 36 h after inoculation.

3.3.Molecular characterization

There were two different sizes of amplicons among all 30 isolates using primer pair ITS4/ITS5 (Table 1),one was approximately 1 000 bp,consistent with inclusion of an intron as previously shown forS.trifoliorum,while the other was about 500 bp,characterstic ofS.sclerotiorumandS.minor(Fig.3).The PCR products of 10 representative isolates were sequenced and the resulting sequences were blasted against the NCBI GenBank Database.Sequence alignments showed that sequences of JH1 and JH3 were 100% identical toS.minor(MN421822);sequences of JH9 and JH13 were 100% identical toS.sclerotiorum(MN216247);and sequences of JH7,JH8,JH16,JH23,JH26,and JH29 were 100% identical toS.trifoliorum(AY547267).The 10 representative isolates were clustered on three clades in phylogenetic tree based on rDNA-ITS sequences,and two isolates formed a distinct clade withS.minor,two isolates withS.sclerotiorum,and six isolates withS.trifoliorum(Fig.4).

Fig.4 Phylogenetic tree of Sclerotinia species constructed using datasets of the rDNA-ITS sequences from this study and GenBank.Bootstrap support values are indicated in the nodes.Monilinia jezoensis were used as an outgroup.

ThreeSclerotiniaspp.specific primer pairs were used to identify the 30 isolates (Table 1).The primer pair SMLcc2F/SMLcc2R specific forS.minoramplified a target product of 264 bp in isolates JH1 to JH6.The primer pair SSasprF/SSasprR specific forS.sclerotiorumamplified a target product of 171 bp in JH9 to JH15.The primer pair STCadF/STCadR specific forS.sclerotiorumamplified in JH7,JH8,and JH16 to JH30 with a target product of 97 bp (Fig.3).

Fig.3 PCR amplification of genomic DNA of 30 isolates using primer pair ITS4/ITS5 and specific primer pairs.M,DNA marker(DL2000).

The molecular characterization confirmed that JH1 to JH6 isolates wereS.minor,JH9 to JH15 isolates wereS.sclerotiorum,and JH7,JH8,and JH16 to JH30 isolates wereS.trifoliorum.

3.4.Pathogenicity tests

All 30 isolates were pathogenic to the pea cultivars Zhongwan 4 and Longwan 1 and caused typical symptoms 3 d after inoculation,while control plants remained healthy (Table 1).The rot stems showed typical symptoms with water-soaked necrotic lesions and were covered with white mold.The necrotic lesions could spread to the adjacent leaves and the base of stems,causing plant stem breaking (Fig.5).The necrotic lesions length ofS.sclerotiorumandS.minorwere similar,with the average size of approximately 6.5 cm,and both were longer than that ofS.trifoliorum(3.1 cm) 3 d after inoculation (Fig.5).The same species as the ones used in inoculation were re-isolated from the inoculated plants,but not from the control plants.

Fig.5 Symptoms caused by three Sclerotinia species on the pea cultivar Zhongwan 4.

4.Discussion

The white mold of pea was first reported in New Zealand,and then this disease has been reported from most pea production areas in the world (Boland and Hall 1994).The pathogens inciting white mold were mainly three species ofSclerotiniaspp.,S.sclerotiorum,S.minorandS.trifoliorum,and they had nearly the same disease cycle and epidemiology (Prattet al.1988;Chenaultet al.2009).Sclerotinia sclerotiorumandS.trifoliorumcould cause similar symptoms such as root,stem,leaves and pods rot,whileS.minorwas reported only to cause root and stem rot in the field (Prattet al.1988;Koikeet al.1996).The disease symptoms on pea observed in Chongqing and Sichuan were similar to the previously described white mold.

In China,Sclerotiniaspp.could cause serious economic losses on some crops every year,especiallyS.sclerotiorumthat was reported almost all over the country.It has been documented to infect more than 150 plant species,and is the main pathogen of rapeseed,peanut,vegetables,sunflower and soybean (Tai 1979;Gaoet al.2009;Ulothet al.2013;Jiaet al.2017).Sclerotinia minorhas been reported on Chinese cabbage,lettuce,peanut and sunflower in Hubei and Inner Mongolia (Lyuet al.2014;Yanget al.2016;Jiaet al.2017).ForS.trifoliorum,it was only reported on alfalfa and milk vetch,in Hubei,Chongqing and Jiangxi (Liet al.1996;Huanget al.2009;Yinet al.2012).

In this study,we conducted field disease surveys of pea.It was found that white mold has become an important disease in some pea cultivation areas of Chongqing and Sichuan.There may be several reasons resulting in the disease severe occurrence on pea,such as the high humidity and cold weather conditions during the pea growing season,adjacent to rape and faba bean fields,incompetent field management including excessive soil nitrogen and heavy seeding rates (Kraft and Pfleger 2001;Jainet al2012).The continuous cropping of the pea,rape,and faba bean susceptible to white mold could aggravate the occurrence of white mold in the fields (Kraft and Pfleger 2001;Porteret al.2009).

The diseased pea plants tissues with typical symptoms and sclerotia were collected from survey fields for pathogen isolation and 30 isolates withSclerotinialike fungi were isolated.Morphological characteristics indicated that 30 isolates could be divided into three differentSclerotiniaspecies according to growth rate and size of sclerotia.Six isolates with moderate growth rate and small sclerotia,seven isolates with fast growth rate and medium sclerotia,and 17 isolates with slow growth rate and big sclerotia,were identified asS.minor,S.sclerotiorum,andS.trifoliorum,respectively (Tariqet al.1985;Njambereet al.2008).

Molecular characterizations have been extensively employed to facilitate accurate species identification in the genusSclerotinia(Baturo-Ciesniewskaet al.2017).Some molecular methods have been proved to be excellent for distinguishingSclerotiniaspecies:Presence or absence of group I introns in the nuclear small-subunit of rDNA (Powerset al.2001),SNP variation in the rDNA intergenic spacer region (Njambereet al.2008),PCR production difference in theβ-tubulingene (Vleugelset al.2012),and the detection of molecular markers specific toSclerotiniaspecies (Abd-Elmagidet al.2013).In this study,molecular phylogenetic analysis based on rDNAITS indicated that the 10 representative isolates formed three distinct clades,clustering withS.minor,sclerotiorumandS.trifoliorum,respectively.The phylogenetic analysis supports the results of morphological characteristics and molecular detection using species-specific primers SMLcc2F/SMLcc2R,SSasprF/SSasprR and STCadF/STCadR respective forS.minor,S.sclerotiorumandS.trifoliorum.

Pathogenicity tests revealed that 30 isolates were highly pathogenic to the pea cultivars Zhongwan 4 and Longwan 1.The typical symptoms appeared on stems with water-soaked necrotic lesions and white mold after inoculation.Moreover,the necrotic lesions caused byS.sclerotiorumwere more serious thanS.trifoliorumin the early stage of infection,likely because it had faster growth rate,which was positively correlated with aggressiveness on young plants (Njambereet al.2008;Vleugelset al.2013).

In China,the earliest record of the occurrence of white mold in pea was in Taiwan of China in 1919,and later the pea disease had been reported in several provinces of China (Tai 1979;Wu 1988).In the previous studies,the pathogen was regarded asS.sclerotiorumbased on morphology (Yang 1959;Wu 1988).In this study,we used morphological characteristics and molecular characterization to identify the pathogens of white mold of pea in Chongqing and Sichuan.In Chongqing,threeSclerotiniaspecies infecting pea were found,while onlyS.trifoliorumwas isolated from Yilong,Sichuan.This is the first report ofS.minorandS.trifoliorumcausing white mold on pea in Chongqing andS.trifoliorumin Sichuan.But due to the limited source and number of samples included in this study,theSclerotiniaspecies causing white mold on pea remain to be assessed.Knowledge on distribution of these three species will be important in developing pea cultivars resistant toSclerotiniawhite mold to disease control.

5.Conclusion

In this study,we isolated 30Sclerotiniaspp.isolates including sixS.minorisolates,sevenS.sclerotiorumisolates and 17S.trifoliorumisolates from Chongqing and Sichuan of China.These pathogens were identified by morphological characters and molecular characterization,and their pathogenicity tests on pea.This study provides important information forSclerotiniaspecies diversity causing white mold on pea in China,which will guide the pea breeding ofSclerotiniaspp.resistance.

Acknowledgements

This study was supported by the China Agriculture Research System of MOF and MARA (CARS-08),the National Crop Germplasm Resources Center of China(NCGRC-2020-09) and the Scientific Innovation Program of the Chinese Academy of Agricultural Sciences.

Declaration of competing interest

The authors declare that they have no conflict of interest.