Isolation and identification of Sclerotinia stem rot causal pathogen in Arabidopsis thaliana§

Isolation and identification of Sclerotinia stem rot causal pathogen in Arabidopsis thaliana § § 1 2 first-author 2 2 2 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yxxc19204@sina.comzonghuaw@163.com †E-mail:yxxc19204@sina.comzonghuaw@163.com yxxc19204@sina.com 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yxxc19204@sina.comzonghuaw@163.com †E-mail:yxxc19204@sina.comzonghuaw@163.com yxxc19204@sina.com Computer and Information College, Fujian Agriculture and Forestry University, Fuzhou 350002, China Computer and Information College, Fujian Agriculture and Forestry University, Fuzhou 350002, China Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China A new stem rot disease is found to occur naturally on Arabidopsis plants in greenhouses of Fuzhou, China. In order to identify its pathogen, we conducted a series of fungal isolation and purification, plant reinoculation, and ascus and ascospore induction from the sclerotia. The isolate caused typical water-soaked lesions after reinoculation and produced sclerotia both on Arabidopsis plants and culture medium plates, and the sclerotia could be induced to produce discal apothecia and 8 binucleate ascospores per ascus. These disease symptom and fungal morphology data revealed that the fungus Sclerotinia sclerotiorum (Lib.) de Bary was the pathogen for Arabidopsis stem rot. To confirm this, we further amplified its large subunit ribosomal DNA (LSU rDNA) by polymerase chain reaction (PCR), and compared the sequence with the known LSU rDNA sequences in GenBank. The results show that the sequence shares the highest identities with the LSU rDNAs of different S . sclerotiorum strains. Taking all these data together, we concluded that the fungus that caused the Arabidopsis stem rot is S . sclerotiorum (Lib.) de Bary. This is the first report that Arabidopsis is naturally infected by S . sclerotiorum . Sclerotinia sclerotiorum Arabidopsis thaliana Large subunit ribosomal DNA (LSU rDNA) Systematic classification INTRODUCTION 1 Sclerotinia sclerotiorum (Lib.) de Bary is a facultative parasitic Ascomycete fungus (Kirk et al., 2001 2001 ), and can grow well even in an unfavorable environment and survive for up to 8 years in soil in the sclerotia form (Adams and Ayers, 1979 1979 ). It can infect as many as 408 plant species including many important crops, such as rapeseeds, sunflower and soybean, and many vegetables (Boland et al., 1994 1994 ; Bolton et al., 2005 2005 ). It causes water-soaked lesions on the leaves or stem rot in stems of some infected plants. The most obvious symptoms of plants infected by S . sclerotiorum are necrotic tissues covered with patches of fluffy white mycelia, and sclerotia are produced after mycelial growth when the nutrition is not sufficient or other conditions are favourable for sclerotial development (Christias and Lockwood, 1973 1973 ). Sclerotia play an important role in disease cycles as they are the primary structures for their long-term survival and produce inocula for further infection (Willetts and Wong, 1980 1980 ). Sizes of sclerotia are dramatically different depending on their host. Sclerotia germinate either carpogenically or myceliogenically, resulting in two distinct categories of diseases under different environmental conditions. Hyphae developed when sclerotia germinate myceliogenically and can directly attack plant tissues under soil. However, apothecia are produced when sclerotia germinate carpogenically and ascospores can be projected to the air and infect aboveground portions of host plants (Bardin and Huang, 2001 2001 ; Le Tourneau, 1979 1979 ; Bolton et al., 2005 2005 ). The large subunit ribosomal DNA (LSU rDNA) has been widely used as a potential marker for fungal species identification in recent years. The LSU is a part of the rDNA gene sequence of the nuclear genome, which is arranged in ribosome clustered at tandem repeat manner (Long and Dawid, 1980 1980 ; Sonnenberg et al., 2007 2007 ). Ribosomal genes are highly conserved in different forms of living organisms (Woese et al., 1990 1990 ), but are actually composed of a mixture of conserved and divergent regions. It is considered that LSU rDNA is an imprint of evolutionary history for different organisms (Lydeard et al., 2000 2000 ). The new disease, with similar symptoms to sclerotia stem rot, was observed when we grew Arabidopsis thaliana in a greenhouse. In order to identify the causal pathogen, we conducted pure isolation and observed the morphology and cytology of the isolate, further sequenced the LSU rDNA and compared the identity with the known S . sclerotiorum LSU rDNA sequences. Our results clarify that the Arabidopsis sclerotia stem rot disease was caused by an isolate of S . sclerotiorum , which will open a new window to study plant- S . sclerotiorum interaction by using the model Arabidopsis plant system. MATERIALS AND METHODS 1 Fungal isolation and purification 2 We followed a standard procedure by Fang ( 1998 1998 ) for fungal isolation and purification. The infected tissues of Arabidopsis were collected from local greenhouses, cut into small pieces, and then rinsed 3~4 times with diluted water after treated with 70% (v/v) ethanol for 2~3 s and with 0.1% (w/v) mercuric chloride solution for 3~5 min. The treated tissues were transferred to potato dextrose agar (PDA) medium and cultured at 25 °C. To confirm pathogenicity of the isolate, the pure culture was reinoculated onto Arabidopsis plants. When actively growing on PDA plates, mycelium-agar plugs were excised from the margin of the fungal colony and used to inoculate Arabidopsis leaves. Inoculated leaves were then incubated in a growth chamber at 23~25 °C with 100% relative humidity until observation. Treatment of sclerotia at low temperature to induce apothecium development 2 The sclerotia produced on PDA medium were collected, placed in moist sands evenly, and then cultured in 4 °C for 6~8 weeks and left in place at 15~20 °C under scattered light until the apothecia developed (Smith and Boland, 1989 1989 ). Microscopic observation of ascospore morphology 2 Apothecia were cut into thin slices and stained with trypan blue solution over night to observe the morphology of asci and ascospores, or stained with 0.5 μg/L ethidium bromide (EB) for 30 min to observe nuclei, or stained with 10 μg/ml Calcofluor white solution for 5 min to observe cell walls and septa between cells, then rinsed with sterile water, and photographed with visible, ultraviolet light and fluorescence under an Olympus microscope, respectively. Polymerase chain reaction (PCR) amplification of LSU rDNA 2 The fungus was cultured by shaking in liquid PDA medium at 25 °C for 3~4 d. Mycelia were collected by centrifugation and its genomic DNA was extracted using a cetyl trimethyl ammonium bromide (CTAB) procedure as described in Talbot et al.( 1993 1993 ) and used as a template for PCR amplification of LSU rDNA. LSU rDNA was amplified with the universal primers NL1 (5′-GCATATCAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) (O′Donnell, 1993 1993 ). The PCR conditions were: 94 °C, 2 min for denature; 33 cycles of 94 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min; and 72 °C, 7 min for extension. The PCR product was separated with 1.5% (w/v) agarose gel and sequenced at Shanghai Ding’an Company, China. Sequence and phylogenetic analysis of LSU rDNA 2 The LSU rDNA sequence of the fungus was sent to National Center for Biotechnology Information (NCBI) ( http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/ ) and analyzed using the basic local alignment search tool (BLAST) sequence comparison algorithms. The high homologous sequences were downloaded and analyzed with program Clustal X. The *.aln files were opened by Mega software and the phylogenetic tree was generated. RESULTS 1 Fungal infection and symptoms 2 The fungal infection process was observed under natural and manual infection. The fungus infected old leaves firstly and expanded rapidly through the petiole into the stem, causing water-soaked lesions. The upper leaves were subsequently infected until the whole plant finally died off. The disease was frequently observed to trans-infect other healthy plants if the infected leaves made contact with uninfected plants (Figs. 1a and 1b 1a and 1b ). The hyphae grew luxuriantly and adjacent plants could also become infected with the disease through the hyphal growth when the atmosphere humidity reached 90% (Fig. 1b 1b ). Sclerotia formed on the surface of plants after plants had died (Fig. 1c 1c ). (a) A primarily infected Arabidopsis plant; (b) Heavily infected Arabidopsis plants; (c) Sclerotia on dead infected plants; (d) Sclerotia starting to develop on PDA plate; (e) Well developed sclerotia on PDA plates. Arrows indicate sclerotia To conduct pathogen identification and analysis, the fungus was isolated from infected tissues of Arabidopsis and cultured on PDA medium. The fungus started to produce white masses when growing to the edge of the Petri dish (Fig. 1d 1d ). The size of mycelium masses became bigger and their colors became darker as time proceeded. Finally, many black sclerotia formed (Fig. 1e 1e ). When the Arabidopsis leaves were inoculated with the isolated fungus, the symptoms were the same as those under natural infection (data not shown). Germination of sclerotia and formation of apothecia 2 To further observe the biology of sclerotia germination and apothecia formation, sclerotia were first incubated in moist sandy soil for 6~8 weeks at 4 °C and then placed in the condition at 15~20 °C with scattered light. Sclerotia geminated quickly after 20 d. Stipes and receptacles of apothecia were formed at first (Fig. 2a 2a ), then their tops grew swollen and discal apothecia with cupped centers were formed (Fig. 2b 2b ). Hollowness in the center became flatter with augmentation of the apothecia and the hymenial layer spread fully until asci became mature and ascospores emanated (Fig. 2c 2c ). The numbers of apothecia produced per sclerotium were not equal, ranging from only 1 to as many as 8~9 apothecia (Fig. 2d 2d ). (a) Primary stage of sclerotia germination; (b) Apothecia being formed; (c) Mature apothecia; (d) Geminated sclerotia with several apothecia formed Morphology of asci and ascospores 2 Apothecia were cut into thin slices, then stained with trypan blue and observed with a microscope. The hymenial layers of the apothecia were found to be full of asci, each ascus containing 8 ascospores. Mature ascospores were released from the top of the asci (Figs. 3a and 3b 3a and 3b ). Many paraphyses could be seen among the asci (Fig. 3c 3c ). Each ascospore had two nuclei when stained by EB (Figs. 3d and 3e 3d and 3e ), but no septum was observed when stained with Calcoflour white (Fig. 3f 3f ). The results suggest that ascospores were single binuclear cells, coinciding with the description in (Kohn, 1979 1979 ). (a), (b) and (c) Slices of apothecia stained with trypan blue: (a) the hymenial layer with rows of asci which are cylindrical sac-like zygote cells, (b) ascus with eight ascospores, (c) paraphysis among asci; (d) and (e) Ascospores stained with EB: (d) observed under visible light, (e) observed under ultraviolet light; (f) Ascospores stained with Calcoflour white observed under florescence LSU rDNA sequence and fungal phylogenetics revealed by the sequence analysis 2 To study phylogenetics of the isolate, we used a pair of universal primers to amplify the LSU rDNA by PCR (O′Donnell, 1993 1993 ). A product with a size of roughly 600 bp was obtained, sequenced and deposited in GenBank (accession No. EU926159). The sequence was analyzed using the BLAST sequence comparison algorithms from the NCBI website ( http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/ ). The analysis revealed that LSU rDNA of our isolate ( S . sclerotiorum strain FZ001) has close evolutionary relationship with fungal species in Discomycetes , including S . sclerotiorum , Botryotinia fuckeliana , Scleromitrula shiraiana , Sclerotinia veratri and Mitrula borealis (Fig. 4 4 ). It was closely clustered in the same group with Sclerotiniaceae members, including S . sclerotiorum , 28S LSU rDNA of S . sclerotiorum strain AFTOL-ID 928, 28S rDNA of S . sclerotiorum strain CBS 499.50, 25S LSU rDNA of S . sclerotiorum strain WZ0067, LSU rDNA of B . fuckeliana and 28S rDNA of B . fuckeliana isolate AFTOL-ID 59 (Fig. 4 4 ). All of these sequences shared more than 99% identities, indicating that they have a notablely tight relationship. B . fuckeliana , the teleomorph of Botrytis cinerea , produces highly resistant sclerotia, but also produces abundant hyaline conidia (asexual spores) borne on grey, branching tree-like conidiophores which is different from S . sclerotiorum (Kirk et al., 2001 2001 ). Other fungi we selected such as S . shiraiana strain Hirayama062001, S . veratri 96CFEBD7, and two M . borealis strains were divergent from this group. These results suggest that our isolate is a strain of S . sclerotiorum . We here named it as S . sclerotiorum strain FZ001. The LSU rDNA and other related sequences were retrieved from GenBank, and calculated with neighbor-joining method. Bootstrap values (%) are indicated at the nodes DISCUSSION 1 The fungus we isolated could infect Arabidopsis , causing water-soaked lesions in leaves, developing sclerotia on the surface of infected tissues and spreading across leaves in contact with each other at high moist conditions. These disease symptoms are the same as that caused by S . sclerotiorum (Lib.) de Bary in other plants. Although some reports indicated that S . sclerotiorum could infect Arabidopsis in manual inoculation, there is no report suggesting that Arabidopsis can be infected naturally. Mycelia of S . sclerotiorum (Lib.) de Bary in host plants or in culture look hyaline, septate, branched and multinucleate, and their colors changed from white to dark as melanin accumulates. It cannot produce conidiophore during the asexual period. Hyphae tend to form sclerotia (Kirk et al., 2001 2001 ). These are also the characteristics of our isolate. Our further observation on sclerotia germination and apothecia production confirms that there are three stages during the course of sclerotial development (Chet and Henis, 1975 1975 ; Bolton et al., 2005 2005 ). At initiation stage, hyphae aggregate to form a white mass, then further aggregate to increase the size of sclerotia; finally, surfaces of sclerotia are delimited, with melanin deposited in peripheral rind cells, and interiors of sclerotia become consolidating. Each sclerotium can produce one or more apothecia consisting of a stipe and a receptacle with a flat to convave hymenial layer (2~10 mm in diameter). Asci are cylindrical sac-like zygote cells and are rowed in the hymenial layer. Each ascus contains eight hyaline, ellipsoid binucleate ascospores [(4~6) μm×(9~14) μm]. The morphology and development of our isolate were similar to those of S . sclerotiorum as described by Kohn ( 1979 1979 ). 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The biology of Sclerotinia sclerotiorum, S. trifoliorum, and S. minor with emphasis on specific nomenclature CR Woese , O Kandler , ML Wheelis . Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya . Proc Natl Acad Sci USA , 1990 . 87 ( 12 ): 4576 - 4579 . DOI: 10.1073/pnas.87.12.4576 http://doi.org/10.1073/pnas.87.12.4576 . Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya 10.1631/jzus.B0860010 Project supported by the National Natural Science Foundation of China (No. 30471178) and the Ministry of Agriculture of China (No. nyhyzx07-054) 2008-08-07 2008-08-25 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 09