| Fruit ring rot, stem warts, and stem canker are important fungal diseases of pear in China. These diseases have led to substantial economic losses due to excessive fruit rots or the severe. For a long time, pear stem wart and stem canker diseases have been considered as two diseases induced by fungi in the genus Botryosphaeria with differences in distribution and symptom. However, the causal agents of pear stem wart and pear stem canker were rarely understood until now, despite the fact that the two diseases are widely present in China. This study was carried out to accurately investigate causal agents of the stem wart and stem canker diseases of pear in China and determine the symptom types of pear diseases caused by those agents. Based on the colony characteristics and dsRNAs extracted from different strains, novel mycoviruses from B. dothidea were biologically and molecularly characterized. The characterization of dsRNA viruses, inclunding structure and characteristics of genome, morphology and structure of VLPs, influence in virulence of host, were detailedly described. The obteinded results should provide useful data for understanding the occurring characteristics of pear stem warts and stem canker and effective biological control of Botryosphaeria diseases using mycoviruses. The results were listed as follows:To identify the species of this genus infecting pear in China, total 243 isolates showing typical culture and conidial morphology of Botryosphaeria spp. on PDA plates were isolated from pear diseased samples from 18 of 20 pear-growing regions. 131 strains were obtained from 129 out of the 243 isolates. Morphological characterization and phylogenetic analyses of the ribosomal DNA internal transcribed spacer region and the β-tubulin and EF1-α genes revealed that B. dothidea, B. rhodina, B. obtusa, and B. parva were associated with different pear stem wart and stem canker symptoms. Botryosphaeria spp. Strains were not isolated from Xinjiang and Gansu province samples, but other samples could obtain the genus fungi. However, there were difference in speices and amounts. For example, B. dothidea was a dominant species(116 out of 131 strains) and distributed in all province. This result showed that B. dothidea had a geographical adaptability. B. rhodina, B. parva and B. obtusa strains were 2, 7 and 6, respectively. Remarkably, all isolates of B. dothidea were obtained from the samples showing either stem wart or stem canker lesions, however, the isolates of the other three species were only obtained from the samples showing stem canker.Pathogenicity tests on the pear shoots showed that B. dothidea isolates could induce stem wart or stem canker lesions but all the isolates of the other three species could only induce stem cankers. The isolates of B. dothidea and B. parva could infect and induce symptoms on unwounded and wounded shoots. The isolates of B. rhodina and B. obtusa could only through infect the wound shoots. However, the isolates of B. parva, B. rhodina, and B. obtusa exhibited higher virulence than that of the B. dothidea isolates on the pear fruit. Our results suggest that B. dothidea is the common causal agent for these two diseases(a pear stem wart and a pear-related stem canker), whereas B. parva, B. rhodina, and B. obtusa only cause pear stem canker diseases. This study represents the first report for biological and molecular characterization of four Botryosphaeria spp. isolated from pear plants showing stem wart and stem canker in China.Based on the colony morphology on PDA, there were seven strains out of 243 strains, which were collected from 16 provinces and two cities, with abnormal colony morphology. The seven strains were classified into three types, four strains grew slowly with abnormal colony morphology which lacked aerial hyphae and turned to dark easily; two strains grew slowly with a normal colony morphology, but had a little of aerial hyphae; one strain grew fast with an abnormal colony morphology without aerial hyphae. Agarose gel electrophoresed analysis of dsRNA isolated from the eight strains showed that there were multiple dsRNA segments. The other strains, 187 strains of B. dothidea, 42 strains of B. parva, 11 strains of B. obtusa and 3 strains of B. rhodina, showed normal colony morphology. On the other hand, extraction the dsRNA from these strains, B. parva, B. rhodina and B. obtusa strains did not isolated dsRNAs. However, dsRNA were isolated from the 95 out of 187 strains of B. dothidea. All strains used in this study were no DNA virus infection.A novel double-stranded RNA(dsRNA) virus, designated as Botryosphaeria dothidea RNA virus 1(BdRV1), isolated from a hypovirulent strain YZN115 of Botryosphaeria dothidea was biologically and molecularly characterized. The genome of BdRV1 comprises of five dsRNAs. Each dsRNA contains a single open reading frame. The proteins encoded by dsRNA1-4 shared significant amino acid identities of 55%, 47%, 43% and 53% with the corresponding proteins of Aspergillus fumigatus tetramycovirus-1. DsRNA1, 3, and 4 of BdRV1 encoded an RNA-dependent RNA polymerase, a viral methyltransferase, and a P-A-S-rich protein, respectively. Function of proteins encoded by the dsRNA2 and dsRNA5 were unknown. DsRNAs and a protein of BdRV1/YZN115 were obtained from virus preparations purified by ultracentrifugation; however, the result of immuuno-capture electron microscopy(ISEM) showed that there were not viral particles decorated by the PAb-p4. BdRV1 conferred hypovirulence for its host and could be transmitted through conidia and hyphae contact.A dsRNA virus was identified from a hypovirulent strain EW220 which was isolated from Hubei. The dsRNA virus was designated as Botryosphaeria dothidea botybirnavirus 1(BdBRV1). The genome of BdBRV1 comprises of two dsRNAs segments with with sizes of 6434 bp and 5986 bp, respectively. Each dsRNA contained a single ORF. ORF1 encoded 1925 amino acids and contained RdRp motifs domain. One large ORF was also found in dsRNA2 which encoded a protein with 1757 amino acid. The function of protein encoded by dsRNA2 was unclear. The 5′-untranslated regions(UTRs) of the coding strands of dsRNAs 1 and 2 were 545 and 567 nt in length. The 3′-UTRs were 115 and 111 nt in length, respectively. The 5′- and 3′- termini of coding strands of the two dsRNAs contained conserved 500 nt and 63 nt sequence, respectively. BLASTp results showed that the proteins encoded by dsRNA1 and 2 shared significant amino acid identities of 82% and 74% with the corresponding proteins of Sclerotinia sclerotiorum botybirnavirus 1(SsBRV1). The hightest indentity of the two viruses was 90% but lowest was 39%. Virus particle of Bd BRV1 is spherical and the virion was 37 nm in diameter. Three structural proteins were identified with masses of 100 kDa, 90 kDa and 80 kDa, respectively. Bd BRV1 conferred hypovirulence for B. dothidea and could be transmitted through conidia.A double-stranded(ds) RNA of 5322 bp in length from strain GY25 of B. dothidea was isolated and sequenced. We have tentatively named Botryosphaeria dothidea victorivirus 1(BdV1). The dsRNA potentially contained two overlapping ORFs. The predicted gene product of ORF1 had the highest amino acid sequence similarity, 37 %(E-value, 8e-101), to the capsid protein of Rosellinia necatrix victorivirus 1(RnV1), and ORF2 had the highest similarity, 44%(E-value, 0.0), to the RdRp of Beauveria bassiana victorivirus NZL/1980(BbV-NZL/1980). Both of these viruses are members of the genus Victorivirus in the family Totiviridae. There was a small ORF at the 5′-terminus whose predicted gene product did not show homology to any known proteins. |
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