In vitro Selection of Strains of Phytophthora cactorum Resistant to Metalaxyl

Isolates of Phytophthora cactorum resistant to the systemic fungicide metalaxyl were obtained by exposing them to sequentially increased concentrations of metalaxyl. A linear relationship was observed between the concentrations of metalaxyl and percentage inhibition of mycelial growth of P. cactorum. The stability of metalaxyl-resistant isolates 150R and 250R was confirmed after six serial transfers on corn meal agar without fungicide. The in vitro metalaxyl-resistant isolate (Ph10) was less aggressive on apple rootstocks compared with the Ph07 isolated from metalaxyl-treated trees and the Ph03 isolated from untreated trees. Metalaxyl-resistant and sensitive isolates remained sensitive to the chemically unrelated fungicide fosetyl-Al at high concentration (600 μg/ml), to mancozeb, and to a mixture of metalaxyl + mancozeb. Significant differences in resistance to metalaxyl existed among P. cactorum field isolates.     

Evaluation of Resistance of Stone Fruit Rootstocks to Phytophthora Crown Rot*

The pathogenicity of Phytophthora citrophthora, Phy‐tophthora cactorum and Phytophthora megasperma strains was confirmed on 2‐year‐old stone fruit rootstocks artificially inoculated in the field. Phytophthora mega‐sperma was not virulent or less virulent than P. cactorum or P. citrophthora. The rootstocks Damas and AN 1/7 were resistant whereas the rootstocks Jl, Titan x Nemaguard 5/1, GF 677, Myrandier 613, Italian x Nemaguard 1 and Myrandier 617 were susceptible to P. citrophthora. The rootstocks Tsukuba 9, Tit‐an x Nemaguard 4/7, AN 1/2, AN 1/3, St. Mien 655/2 and Bl showed medium susceptibility to this species. The isolates of P. cactorum induced more extensive necrosis in Myrandier 617 and Italian x Nemaguard than in Myr‐andier 613, GF 677, Jl (ADAFUEL), Tsukuba 9 and St. Julien 655/2. Among seven rootstocks, Tsukuba 5 and St. Julien 655/2 were more resistant than Myrandier 613, Myrandier 617, Jl (ADAFUEL), Tsukuba 9 and St. Julien 655/2.     

Evaluation of fatty acid methyl ester profile and amplified fragment length polymorphism analyses to distinguish five species of Phytophthora associated with ornamental plants

Fatty acid methyl ester (FAME) profiles and amplified fragment length polymorphisms (AFLPs) were evaluated as tools for identifying species of Phytophthora. Five isolates of each of Phytophthora cactorum, Phytophthora citrophthora, Phytophthora cinnamomi, Phytophthora nicotianae and Phytophthora cryptogea were subjected to both analyses to examine variation among and within species. In FAME analysis, isolates of P. cactorum, P. cinnamomi and P.nicotianae were clustered by species, but isolates of P. citrophthora and P.cryptogea were divided into multiple clusters based on greater variations within these two species. The AFLP analysis differentiated all five species of Phytophthora. The five isolates of each species were grouped in a separate terminal cluster, but diversity within a species cluster varied considerably with variation greater in P. cryptogea and P. citrophthora. Comparing the dendrograms based on FAME and AFLP analyses, the overall patterns of both were similar. The P. cactorum cluster was distinct from clusters of the other four species, which formed one large cluster. The higher values of percentages of polymorphic loci and gene diversity in AFLP analysis substantiated diversity observed among isolates of P. citrophthora and P. cryptogea in FAME and AFLP dendrograms. Both FAME and AFLP appear to be useful tools for identifying species of Phytophthora, but only AFLP analysis has potential to study genetic and phylogenetic relationships within and among species in this genus.     

核糖体基因ITS作为苎麻疫霉、恶疫霉分类辅助性状的研究*

以2个雄器大多围生、少数侧生的苎麻疫霉菌株与1个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ITS）通用引物,PCR扩增3个供试菌株核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果是苎麻疫霉的ITS1和ITS2分别由206和453个碱基组成, 而恶疫霉则分别由218和415个碱基组成。2个供试苎麻疫霉菌株的ITS1和ITS2的碱基序列同源性均分别为100%。苎麻疫霉和恶疫霉ITS1同源性为74.9%,其中中间区域40bp-164bp之间在两种间变异丰富,同源性只有59.4%,而1bp-39bp和165bp-239bp两区域的同源性分别为92.3%和92.1%; ITS2在两种疫霉菌间的同源性为71.0%。结果表明苎麻疫霉和恶疫霉ITS的碱基序列有明显差异。上述结果提示,ITS区域碱基序列可区分苎麻疫霉和恶疫霉。     

核糖体基因ITS作为苎麻疫霉、恶疫霉分类辅助性状的研究

以2个雄器大多围生、少数侧生的苎麻疫霉菌株与1个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ITS）通用引物,PCR扩增3个供试菌株核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果是苎麻疫霉的ITS1和ITS2分别由206和453个碱基组成,而恶疫霉则分别由218和415个碱基组成。2个供试苎麻疫霉菌株的ITS1和ITS2的碱基序列同源性均分别为100%。苎麻疫霉和恶疫霉ITS1同源性为74.9%,其中中间区域40bp-164bp之间在两种间变异丰富,同源性只有59.4%,而1bp-39bp和165bp-239bp两区域的同源性分别为92.3%和92.1%;ITS2在两种疫霉菌间的同源性为71.0%。结果表明苎麻疫霉和恶疫霉ITS的碱基序列有明显差异。上述结果提示,ITS区域碱基序列可区分苎麻疫霉和恶疫霉。     

Complete Genome Sequence of a Novel Wild tomato mosaic virus Isolate Infecting Nicotiana tabacum in China

Virus particles of approximately 740–760 nm in length and 13 nm in diameter were observed from a diseased Nicotiana tabacum (tobacco) plant in Sichuan Province, China. The complete genomic sequence of the virus isolate XC1 was determined to contain 9659 nucleotides without 3′ terminal poly(A) tail. XC1 has a genome typical of members of the genus Potyvirus, encoding a large polyprotein of 3075 amino acids. Putative proteolytic cleavage sites and a number of well characterized functional motifs were identified by sequence comparisons with those of known potyviruses. Sequence comparison revealed that XC1 shared the highest level of nucleotide sequence identity (76.5%) with Wild tomato mosaic virus (WTMV). Phylogenetic analysis showed that XC1 was closely related to the WTMV Guangdong isolate with an identity of 94.3% between CP gene sequence of the two viruses. We thus named XC1 WTMV‐XC‐1 as a novel isolate of WTMV. The full sequence of WTMV‐XC‐1 may serve as a basis for future investigations on the gene diversity of WTMV.     

Effect of Bacterial Antagonist on Phytopthora cactorum and Apple Crown Rot

Bacterial antagonist B8 produced an inhibition zone with each of four Phytophthora cactorum isolates on corn meal agar (CMA) plates. Infections with three P. cactorum isolates were significantly reduced when the soil was simultaneously inoculated with B8. Growth of P. cactorum was completely inhibited on CMA amended with 40–100 per cent 10 fold concentrated B8 extract. Percent oospore germination of P. cactorum was significantly reduced when B8 was present in suspension for 9, 12 and 15 days from inoculation. Survival of oospores was significantly reduced at 60 and 90 mm depths in soil. Bacterial antagonist B8 significantly reduced the population of viable P. cactorum oospores in the top 30 mm of soil where oospores generally survive.     

Use of Cross-Reactive Antibodies to Detect Members of the Potyviridae

In attempts to use cross-reacting antibodies for the broad-spectrum detection of potyviruses two broad-spectrum immunoreagents, the monoclonal antibody P-3-3H8 and antiserum Tu MV-314 raised against an isolate of peanut stripe virus and turnip mosaic virus, respectively, were examined for their ability to detect members of the Potyviridae in a simple indirect plate-trapped antigen (PTA) ELISA. Both immuno-reagents reacted strongly not only with isolate of 50 different virus species of the genus Potyvirus but also with several isolates of ryegrass mosaic virus, the type member of the genus Rymovirus A few members of the genus Potyvirus as well as agropyton mosaic and hordeum mosaic viruses, two other species of the genus, Rymovirus did not react with P-3-3H8 but only with TuMV-314 which showed the highest degree of cross-reactivity. In no case were positive reactions obtained with members of the genera Bymovirus and Ipomovirus. These two immunoreagents which appear to be directed to conserved epitopes gave good results when they were employed for detecting potyviruses in field samples from ornamental (Liliales) and leguminous plants. The potential and limitations of cross-reactive antibodies for the routine detection of potyviruses are discussed.     

传染性法氏囊病毒的抗原及分子特征

用鸡胚成纤维细胞对来自野外的 5 个传染性法氏囊病毒株 (IBDV-JD1 、 JD2 、 NB 、 HZ1 、 HZ2) 进行分离,测定理化特性、致病性,同时进行血清亚型测定及 A 片段基因组的克隆分析 . 试验所用 5 个法氏囊组织悬液在鸡胚成纤维细胞盲传 2~14 代后适应细胞并产生细胞病变 . 细胞适应的 IBDV 毒株的理化和形态特征与经典传染性法氏囊病毒株一致 . 除 IBDV-HZ1 、 HZ2 属经典 IBDV 血清型外, IBDV-JD1 、 JD2 和 NB 毒株分属不同的血清亚型 . 人工感染实验结果显示,分离的 IBDV 毒株产生与野外病例相似的临床症状和病变,出现法氏囊滤泡髓质的淋巴细胞变性、坏死和消失 . 基因组序列分析显示, IBDV-NB 毒株 A 片段由 3 264 个核苷酸组成,编码由 145 个氨基酸残基组成的 VP5 和由 1 012 个氨基酸残基组成的多聚蛋白 . 与来自 GenBank 的 IBDV Ⅰ型毒株比较, NB 毒株 A 片段编码的多聚蛋白与 JD1 毒株的同源性最高,达 99.5% , VP2 与 JD1 、 CEF94 、 D78 的同源性为 99.8% , VP3 与 JD1 的同源性为 99.2% , VP4 与 JD1 的同源性为 100% , VP5 与 JD1 , HZ2 , P2 , CEF94 , CT , Cu-1 和 D78 毒株的同源性为 99.3%. NB 毒株 VP2 蛋白的第 253 、 280 、 284 位氨基酸残基与 IBDV 变异毒株和经典毒株一致,但不同于 IBDV 超强毒株 . 这些结果暗示 IBDV 的抗原表位是构象依赖性表位, IBDV 血清亚型的形成与 IBDV 弱毒疫苗病毒株密切相关 .     

The apparent non‐host resistance of Ethiopian mustard to a radish‐infecting strain of Turnip mosaic virus is largely determined by the C‐terminal region of the P3 viral protein

Two different isolates of Turnip mosaic virus (TuMV: UK 1 and JPN 1) belonging to different virus strains were tested on three different Brassica species, namely turnip (Brassica rapa L.), Indian mustard (Brassica juncea L.) and Ethiopian mustard (Brassica carinata A. Braun). Although all three hosts were readily infected by isolate UK 1, isolate JPN 1 was able to establish a visible systemic infection only in the first two. Ethiopian mustard plants showed no local or systemic symptoms, and no virus antigens could be detected by enzyme‐linked immunosorbent assay (ELISA). Thus, this species looks like a non‐host for JPN 1, an apparent situation of non‐host resistance (NHR). Through an experimental approach involving chimeric viruses made by gene interchange between two infectious clones of both virus isolates, the genomic region encoding the C‐terminal domain of viral protein P3 was found to bear the resistance determinant, excluding any involvement of the viral fusion proteins P3N‐PIPO and P3N‐ALT in the resistance. A further determinant refinement identified two adjacent positions (1099 and 1100 of the viral polyprotein) as the main determinants of resistance. Green fluorescent protein (GFP)‐tagged viruses showed that the resistance of Ethiopian mustard to isolate JPN 1 is only apparent, as virus‐induced fluorescence could be found in discrete areas of both inoculated and non‐inoculated leaves. In comparison with other plant–virus combinations of extreme resistance, we propose that Ethiopian mustard shows an apparent NHR to TuMV JPN 1, but not complete immunity or extreme resistance.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

The Feasibility of Using High Resolution Genome Sequencing of Influenza A Viruses to Detect Mixed Infections and Quasispecies

Background

The rapidly expanding availability of de novo sequencing technologies can greatly facilitate efforts to monitor the relatively high mutation rates of influenza A viruses and the detection of quasispecies. Both the mutation rates and the lineages of influenza A viruses are likely to play an important role in the natural history of these viruses and the emergence of phenotypically and antigenically distinct strains.

Methodology and Principal Findings

We evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs (six waterfowl isolates - five H3N2 and one H4N6; an H7N3 turkey isolate; and a bald eagle isolate with H1N1/H2N1 mixed infection), and two tissue cultured H3N2 swine influenza viruses. Two waterfowl cloacal swabs were included in the analysis. Full-length sequences of all segments were obtained with 20 to 787-X coverage for the ten viruses and one cloacal swab. The second cloacal swab yielded 15 influenza reads of ∼230 bases, sufficient for bioinformatic inference of mixed infections or quasispecies. Genomic subpopulations or quasispecies of viruses were identified in four egg grown avian influenza isolates and one cell cultured swine virus. A bald eagle isolate and the second cloacal swab showed evidence of mixed infections with two (H1 and H2) and three (H1, H3, and H4) HA subtypes, respectively. Multiple sequence differences were identified between cloacal swab and the virus recovered using embryonated chicken eggs.

Conclusions

We describe a new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs and add to the understanding of the ecology of influenza A virus populations.     

Suppression of Phytophthora blight on pepper (Capsicum annuum L.) by bacilli isolated from brackish environment

Bacteria of the genus Bacillus are well known to possess antagonistic activity against numerous plant pathogens. In the present study, 11 strains of Bacillus spp. were isolated from a brackish environment and assayed for biocontrol activity under in vitro and in vivo conditions. Among the 11 isolates tested, nine isolates effectively inhibited the growth of various plant pathogens, namely Phytophthora capsici, Phytophthora citrophthora, Phytophthora citricola, Phytophthora sojae, Colletotrichum coccodes, Colletotrichum gloeosporioides, Colletotrichum acutatum, Rhizoctonia solani, Fusarium solani, Fusarium graminearum, Pyricularia spp., and Monilina spp. The effective isolates were further screened for suppression of Phytophthora blight of pepper plants under greenhouse conditions. The isolate SB10 exhibited the maximum (72.2%) ability to reduce the disease incidence and increased (32.2%) the vigour index of Capsicum annuum L. plants. Antifungal compounds produced by isolate SB10 were highly thermostable (100°C for 30 min). Matrix-Assisted Laser Desorption Ionization-Time of Flight mass spectrometry of the antifungal compounds revealed three lipopeptide complexes, namely the surfactins, the iturins, and the fengycins, which are well-known antifungal compounds produced by Bacillus spp.     

Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization

Background

Porcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).

Results

Five genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.

Conclusions

These results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.     

The isolation and identification of rhubarb viruses occurring in Britain

Virus-like symptoms were common in British crops of rhubarb. All plants tested of the three main varieties, ‘Timperley Early’, ‘Prince Albert’ and ‘Victoria’, were virus-infected. Turnip mosaic virus and a severe isolate of arabis mosaic virus (AMV) were obtained from ‘Timperley Early’; and ‘Prince Albert’ contained turnip mosaic virus, cherry leaf roll virus (CLRV), a mild isolate of AMV and, infrequently, cucumber mosaic virus (CMV). The main commercial variety ‘Victoria’ contained turnip mosaic virus, CLRV, a mild isolate of AMV and, infrequently, strawberry latent ringspot virus (SLRV). All the viruses were identified serologically. The rhubarb isolates did not differ markedly from other isolates of these viruses in herbaceous host reactions, properties in vitro or particle size and shape. A rhubarb isolate of CLRV was distinguished serologically from a cherry isolate of the virus. Turnip mosaic virus, CLRV and SLRV, were transmitted with difficulty, but AMV isolates were readily transmitted by mechanical inoculation. Turnip mosaic virus was also transmitted to rhubarb by Myzus persicae and Aphis fabae. CLRV was transmitted in 6–8% of the seed of infected ‘Prince Albert’ and ‘Victoria’ rhubarb and in 72% of the seed of infected Chenopodium amaranticolor. Mild isolates of AMV were also transmitted in 10–24% of the seed of infected ‘Prince Albert’ and ‘Victoria’ plants.     

Identification of viruses isolated from plum trees affected by decline, line-pattern and ringspot diseases

Viruses obtained from plum trees infected with either decline, line-pattern or ringspot diseases were characterized and identified. All the viruses were serologically related to Prunus necrotic ringspot virus (NRSV); those from trees with decline or ringspot were serologically indistinguishable from cherry strains of NRSV but differed in pathogenicity, whereas the virus from trees with line pattern was closely related to apple mosaic virus. When returned from herbaceous hosts to Prunus, line-pattern and ringspot isolates reproduced symptoms characteristic of the diseased sources. One virus isolate from trees with decline diminished the vigour of young plum trees. Comparison with other investigations shows that at least two unrelated viruses cause plum line-pattern disease in America and Europe.     

甘蔗花叶病毒3’末端基因的克隆及外壳蛋白序列分析比较

选取我国SCMV优势株系A株系的分离物SCMV-CA为材料,经过病毒和病毒RNA的提纯,反转录获得病毒cDNA,并克隆到载体pUC19的SmaI位点上,筛选得到多个重组质粒。选取其中一个克隆SCMV-CA54进行测序,得到一个全长为1296 bp的核苷酸序列。这段序列由一个长为1044 bp的开放阅读框架（ORF）和一个长279 bp的3’末端非编码区序列（3'-UTR）及poly(A)尾巴组成。这个ORF包括病毒完整的外壳蛋白（CP）及部分核内含体蛋白b（NIb）基因序列。将所得序列同已知SCMV亚组中各株系分离物的核苷酸和氨基酸进行同源性比较,结果表明该序列与其它株系分离物CP核苷酸序列的同源性介于63.7%～77.6%之间,氨基酸的同源性介于64%～89%之间。根据马铃薯Y病毒属的序列同源性划分标准,SCMV-CA与其它株系或分离物的同源性关系均介于种与株系划分标准之间。这是我国首次报道SCMVCP基因序列。     

Sources of crown rot (Phytophthora cactorum) infection in strawberry and the effect of cold storage on susceptibility to the disease

Cold-stored plants of strawberry cultivars Tamella, Cambridge Favourite and Redgauntlet were more susceptible to pathogenic isolates of Phytophthora cactorum than similar plants which had not been cold-stored. Indigenous nonpathogenic isolates of P. cactorum did not cause crown rot in cold-stored plants, although a small number of symptomless latent infections occurred. The majority of P. cactorum isolates causing crown rot symptoms were taken from infected strawberry crowns, although two isolates from gooseberry plants, but of uncertain origin, were also pathogenic. Outbreaks of crown rot in areas with no previous history of the disease therefore probably result from the importation of non-indigenous inoculum with planting material. Assessments of the timing of infection in relation to cold storage revealed that a high incidence of death in the cold store and chronic wilt symptoms on planting from the store resulted from initiating symptomless infections prior to cold storage. However, infection during the period immediately after cold storage resulted in rapid wilt symptoms of Phytophthora crown rot. When plated in sterile distilled water for 24 h, pieces of tissue from infected plants which had died during cold storage produced large numbers of sporangia and zoospores. This indicates that such plant material could provide a potent source of inoculum for infections in the post storage thawing environment. It is proposed that a combination of heightened host susceptibility resulting from cold storage and the presence of scatted latent infections or infected debris among the plants could result in a sudden, large scale appearance of crown rot, as sometimes is seen with cold-stored plantings of strawberries.