禾谷镰孢菌对多菌灵的敏感性基线及抗药性菌株生物学性质研究

 引起小麦赤霉病的禾谷镰孢菌(Fusarium graminearum)在华东地区对多菌灵已出现了高水平抗药性。本文报道了F.graminearum对多菌灵的敏感性基线及其抗药性菌株生物学特性。离体条件下多菌灵对100个野生敏感型菌株的平均EC₅₀和MIC值分别为0.5748±0.0133 μg/ml和 < 1.4μg/ml。而对50个抗药性菌株的平均EC₅₀和MIC值则分别为9.2375μg/ml和 > 100 μg/ml。在麦穗上多菌灵对50个敏感菌株和抗药性菌株防效的平均EC₅₀分别为282.6 μg/ml和 > 2 000μg/ml。从田间获得的抗多菌灵菌株对苯菌灵、噻菌灵、甲基托布津表现交互抗药性,但不同于室内突变菌株,对乙霉威不表现负交互抗药性。抗药性菌株的无性和有性繁殖后代以及在无药培养基上菌丝体转代培养后,抗药水平保持不变。抗药性菌株的菌丝生长速率、产孢能力及致病力等与敏感菌株相比没有差异。     

棉铃疫病菌(Phytophthora boeh meriae)对甲霜灵的抗性遗传研究

 本文在实验室条件下,经药物直接诱变,筛选获得棉铃疫病菌(Phytophthora boehmeriae Sawada)抗甲霜灵突变株,研究了其抗药性性状的遗传。结果表明,棉铃疫病菌易对甲霜灵产生抗性,抗性菌株对甲霜灵的抗性水平可高达其敏感亲本的5161.3倍以上;部分抗性菌株的抗药性性状在无性及有性单孢后代均可稳定遗传,另有部分抗性菌株的抗性性状在无性单孢后代出现分离甚至完全丧失。初步认为,供试棉铃疫病菌对甲霜灵的抗性遗传可能与细胞质遗传因子有关     

恶疫霉有性生殖交配行为的研究

 对同宗配合疫霉种——恶疫霉(Phytophthora cactorum Schroter)抗甲霜灵突变株和抗地茂散突变株的抗药性在游动孢子和卵孢子后代的遗传分析,筛选到抗性分别由细胞核纯合显性单基因控制的抗甲霜灵突变株和由细胞质可稳定遗传的线粒体基因控制的抗地茂散突变株。将携带抗甲霜灵对地茂散敏感(即Mt^rCn^s)标记的亲本与携带抗地茂散对甲霜灵敏感(即Mt^sCn^r)标记的亲本共同培养,在200个有性生殖单卵孢后代中检测到携带3种不同抗药性标记的单孢株,其中32株携带Mt^rCn^s标记,165株携带Mt^sCn^r标记,另有3株同时携带双亲的标记性状即MtrCnr。结果表明:恶疫霉种内不同菌株共同培养时,有性生殖以配对亲本的各自自交为主,同时配对亲本间也可发生一定频率的杂交。     

恶疫霉有性杂交后代的生物学研究

 将2个带有不同抗药性标记的恶疫霉菌株配对,培养36 d后诱导其卵孢子萌发,从3760个单卵孢株中,获得50株带有双亲标记的杂交个体。对其中32株杂交个体的生长速率、有性和无性繁殖能力、卵孢子和游动孢子萌发率、致病力及对高温的耐受力等生物学性状进行测定。结果显示,测试的32株杂交个体在LBA培养基上均能较好地生长,有24株的杂交个体的生长速率介于2亲本之间;在LBA和SL培养基上,大多数杂交个体均能产生较大数量的卵孢子,有37%的杂交个体在SL培养基上产卵孢子能力显著大于双亲,仅有1个杂交个体不产生卵孢子;在人工培养条件下,大多数供试杂交个体可以产生较大数量的孢子囊,其中15株杂交个体产孢子囊能力处于双亲之间;被测的杂交个体产生的卵孢子或游动孢子均可以萌发形成有效的单孢株,有22株的杂交个体的卵孢子萌发率大于50%;被测的杂交个体接种在苹果上都有较强的致病力,有16株杂交个体的致病力显著大于双亲。表明在群体水平上恶疫霉有性杂交后代具有较强的生活能力,提示同宗配合恶疫霉不同菌株间的有性重组,对该种的群体遗传多样性可能具重要作用。     

紫外-氯化锂复合诱变哈茨木霉产生多菌灵抗药性菌株的研究

多菌灵是植物病害防治中常用的化学杀菌剂,野生型哈茨木霉对多菌灵比较敏感。为了能够更好地将哈茨木霉菌应用于实践,本试验采用紫外线-氯化锂复合诱变的方法,实现哈茨木霉菌株对多菌灵的抗性改良。试验共获得315株正向突变菌株,其中hcb-35菌株抗性能力较强。利用毒力测定方法检测了多菌灵对hcb-35有效抑菌中浓度,及hcb-35对多菌灵的抗药遗传稳定性;并利用对峙试验及显微观察检测其抑菌能力。结果显示,与哈茨木霉出发菌株hc相比,多菌灵对哈茨木霉突变株hcb-35菌株的有效抑菌中浓度提升285%;连续转接12代后,hcb-35菌株抗药性相对稳定且抑菌效果较出发菌株无明显差异,表明应用紫外-氯化锂复合诱变哈茨木霉可以获得遗传稳定的耐多菌灵突变株。     

辣椒疫霉产毒缺陷与抗药性突变体筛选及其遗传特性

采用MNNG诱变与自交（S1－S3）纯合的方法,从辣椒疫霉的游动孢子群体中,筛选出1株带有抗霜脲氰标记的产毒缺陷突变体、2株抗甲霜灵的产毒突变体。产毒缺陷菌体和产毒菌株均对茄门甜椒致病,但产毒缺陷菌株的致病力降低一半,证明辣椒疫霉的致病显症过程与毒素作用有关,病菌毒素是重要的致病因子。此外,突变菌株的抗药性和产毒缺陷表型在无性游动孢子后代和有性孵孢子后代群体中均可稳定遗传。在姐妹配对F2代卵孢子群体中,辣椒疫霉毒素产生和对甲霜灵的抗性为不完全显性基因所控制,而对霜脲氰的抗性为完全显性基因所控制。     

玉蜀黍赤霉对小麦品种致病力的测定方法和致病力的分化

 玉蜀黍赤霉[Gibberella zeae(Schw) Petch]的子囊孢子、分生孢子和菌丝体对麦穗的致病力相同。以1-2个子囊孢子或分生孢子接种麦穗即能引起穗腐,将孢子的接种量增加到10个以上,侵染效率也能达100%。菌丝体接种的侵染速度要比孢子快。小麦扬花期离体接种小穗,记载病原菌侵入小穗轴和主穗轴的程度,是鉴定小麦品种抗扩展的可靠方法。
根据对在小麦感病品种矮秆早或抗病品种苏麦三号小穗接种测定的结果,全国各地具有代表性的56个菌株的致病力强弱不同。从强菌株F69分离到的37个单子囊孢子菌株其致病力有显著差异,但致病力的强弱不稳定。从同一个子囊分离到的单子囊孢子菌株,它们的致病力也有分化。菌株的致病力虽然有不稳定性,但不同品种的抗性还是明显的。试验证明玉蜀黍赤霉是同宗配合的,因此关于它的变异问题有待从各个方面进行深入研究。     

禾谷镰孢菌γ-微管蛋白基因克隆及其与多菌灵抗药性关系分析

 根据禾谷镰孢菌参考菌株NRRL31084(PH-1)的γ-微管蛋白基因核苷酸序列设计5对引物,采用PCR方法从禾谷镰孢菌(Fusarium graminearum)对多菌灵(MBC)的敏感菌株、室内诱导及田间多菌灵抗药性菌株中分段扩增测序,获得了γ-微管蛋白基因全序列。该基因全长1 868bp,含有5个内元,编码一含493aa的多肽;与PH 1的γ-微管蛋白基因核苷酸序列同源性99%,存在10个差异核苷酸,与所编码的氨基酸序列同源性100%;与其它7种真菌的γ-微管蛋白基因所编码的氨基酸序列同源性为31%~72%。中国的2个敏感菌株和4个抗药菌株的γ-微管蛋白基因序列完全相同,认为多菌灵抗药性与该微管蛋白变异无关。     

辣椒疫霉对烯酰吗啉的敏感性基线及室内抗药突变体研究

 采用菌丝生长速率法测定了125株采自河北、内蒙古、陕西、安徽和北京等地区的辣椒疫霉病菌对烯酰吗啉的敏感性,结果表明,其EC₅₀值分布于0.126~0.318μg/mL之间,最不敏感菌株是最敏感菌株的2.5倍,平均EC₅₀=(0.218±0.0368)μg/mL。125个菌株对烯酰吗啉的敏感性分布呈单峰曲线,未出现抗性的病原菌亚群体,可将该单峰曲线作为辣椒疫霉对烯酰吗啉的敏感性基线,将烯酰吗啉对该病原群体的平均EC₅₀值作为田间抗药性监测的参考标准。通过紫外诱变敏感菌株N-7的菌丝获得了12株抗烯酰吗啉的突变体,抗性指数在16.38~132.15之间;通过紫外诱变敏感菌株DZ-16的游动孢子获得1株抗性指数为680倍的高抗药水平的突变体。突变体的部分生物学性状研究表明,其致病力与敏感菌株相当;大部分突变体产生孢子囊的能力与亲本菌株相比均有不同程度的提高;离体条件下,突变体和亲本菌株释放游动孢子的能力相当;突变体的菌丝生长速率与亲本菌株相比具有不同程度的差异。测定了敏感亲本菌株和突变体的交配型,均为A1型菌株,经紫外诱变后交配型没有发生改变。综合分析表明,抗性突变体的产生有利于抗药性群体的发展。为避免和延缓抗药性的产生,生产上应将烯酰吗啉与其它无交互抗药性的杀菌剂交替使用。     

浙江衢州地区柑橘绿霉病菌对抑霉唑和多菌灵的抗性水平及其分子机制

研究了采自浙江衢州地区,包括柯城区、衢江区和开化县12个贮藏库的70个柑橘绿霉病菌Penicillium digitatum菌株对抑霉唑和多菌灵的抗性频率、抗性水平及其抗性分子机制。结果表明:柯城区和衢江区的抑霉唑抗性菌株(最低抑制浓度MIC≥0.5 μg/mL)的比例分别为77.1%和62.5%,两地抗性菌株的平均EC50值分别为2.07±1.04 μg/mL和2.35±0.73 μg/mL,分别是当地敏感菌株EC50值的41.4和47.0倍;而采自开化县的菌株均对抑霉唑敏感(MIC≤0.1 μg/mL),平均EC50值为0.04±0.02 μg/mL。柯城区和衢江区的多菌灵抗性菌株(MIC≥10 μg/mL) 的比例分别为54.3%和54.2%,而开化县的抗性菌株比例仅为9.1%。即来自柯城和衢江两个柑橘主产区的绿霉病菌群体对抑霉唑和多菌灵的抗性频率均远高于非柑橘主产区的开化县群体,说明抗药性群体的形成与药剂使用历史有关。进一步研究发现,衢州地区柑橘绿霉病菌对抑霉唑的抗性均属于IMZ-R3型,即与抑霉唑靶标基因 CYP51B 启动子区的插入突变有关,而对多菌灵的抗性则与 β-微管蛋白基因的992位核苷酸点突变(T→A)导致对应的200位点的氨基酸突变(F→Y)有关。     

Vegetative Compatibility of Fusarium graminearum Isolates and Genetic Study on Their Carbendazim-Resistance Recombination in China

ABSTRACT Monoconidial isolates of 33 carbendazim-sensitive isolates and 31 carbendazim-resistant isolates of Fusarium graminearum were selected from three regions of China for vegetative compatibility group (VCG) analysis. A total of 213 and 224 nit mutants were recovered from the 33 sensitive and the 31 resistant isolates, respectively. Of all the nit mutants, the frequency of the different phenotypes was 44.6, 46.5, 5.7, and 3.2% for nit1, nit3, nitM, and nitA, respectively. VCG analysis identified 30 different VCGs among the 33 sensitive- and the 31 carbendazim-resistant isolates, with VCG diversity 0.91 and 0.97, respectively. Both, a carbendazim-sensitive and a -resistant isolate from the same field belonged to the same VCG. In all then, a total of 59 VCGs were identified among the 64 isolates with an overall VCG diversity 0.92. Direct hyphal fusion was observed in six pairs of vegetatively compatible complements, which is evidence of heterokaryon formation. It was hypothesized that carbendazim resistance could not be transferred by hyphal fusion or there is a small chance to be transferred between two compatible isolates. Three stable sexual recombinants of F. graminearum were randomly chosen from each of the three genetic crosses to study their biological properties. There were no significant differences in mycelial linear growth and pathogenicity between recombinants and their parents, but they differ in sporulation ability and capacity to produce perithecia. We concluded that sexual recombination presumably played a role in the development of carbendazim resistance under field conditions.     

Monogenic resistance to a new fungicide, JS399-19, in Gibberella zeae

The fungicide JS399-19 is a novel cyanoacrylate fungicide active against Gibberella zeae , and has been marketed in China for control of fusarium head blight (FHB) on wheat. Forty-three isolates sensitive to fungicide JS399-19 were collected from three commercial wheat fields in China. Forty-five isolates resistant to JS399-19, obtained from five sensitive isolates by selection for resistance to JS399-19, were selected. Three sensitivity levels were identified: sensitive (S), moderately resistant (MR) and highly resistant (HR) to JS399-19, based on a previous study. Eight isolates representing the three sensitivity-level phenotypes were randomly selected for a study on the inheritance of JS399-19 resistance by analysing the sensitivity of hybrid F₁ progeny. A nitrate-non-utilizing mutant ( nit ) was used as a genetic marker to confirm that individual perithecia were the result of outcrossing. Five crosses were assessed: S × S, S × HR, MR × HR, HR × HR and MR × S. In crosses between parents with different sensitivity levels, such as S × HR, MR × HR and MR × S, the progeny fitted a 1:1 segregation ratio of the two parental phenotypes. No segregation was observed in the crosses S × S and HR × HR. It was concluded that the MR and HR phenotypes in G. zeae were conferred by different allelic mutations within the same locus. In these isolates, resistance to JS399-19 was not affected by modifying genes or cytoplasmic components.     

Sexual recombination of carbendazim resistance in Fusarium graminearum under field conditions

BACKGROUND: Carbendazim has been the major fungicide for control of Fusarium head blight (FHB) caused by Fusarium graminearum Schwade in China. However, the effectiveness of carbendazim has been threatened by the emergence of resistant pathogen populations in the field. RESULTS: Five isolates, representing three phenotypes of different carbendazim sensitivity levels (S, MR and HR), were randomly selected to study the inheritance of carbendazim resistance by three genetic crosses under field conditions. Each parent in all crosses was marked with a different class of nitrate non‐utilizing (nit) mutation. The presence of sexual recombinants indicated that outcrossing had occurred in the crosses. Over 100 putative self‐crossing or outcrossing perithecia for each cross were randomly sampled on the surface of the haulms of dead rice for each pair of parents. Results showed that 5.7–20.9% outcrossing frequency occurred in the three crosses and confirmed sexual recombination under field conditions. There were no significant differences in mycelial linear growth and pathogenicity between the selected recombinants and their parents, but they differed in sporulation ability and capacity to produce perithecia. Nevertheless, most of the sexual recombinants possessed fitness levels comparable with those of their parents. CONCLUSION: Outcrossing between carbendazim‐sensitive and ‐resistant isolates did occur under field conditions, and sexual recombination must play a role in the development of carbendazim resistance in the field. Copyright © 2009 Society of Chemical Industry     

Sexual Recombination in Gibberella zeae

ABSTRACT We developed a method for inducing sexual outcrosses in the homothallic Ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum). Strains were marked with different nitrate nonutilizing (nit) mutations, and vegetative compatibility groups served as additional markers in some crosses. Strains with complementary nit mutations were cocultured on carrot agar plates. Ascospores from individual perithecia were plated on a minimal medium (MM) containing nitrate as the sole nitrogen source. Crosses between different nit mutants segregated in expected ratios (3:1 nit(-):nit(+)) from heterozygous perithecia. Analysis of vegetative compatibility groups of progeny of two crosses indicated two and three vegetative incompatibility (vic) genes segregating, respectively. For rapid testing of sexual recombination between nit mutants, perithecia were inverted over MM to deposit actively discharged ascospores. Development of proto-trophic wild-type colonies was taken as evidence of sexual recombination. Strains of G. zeae group 2 from Japan, Nepal, and South Africa, and from Indiana, Kansas, and Ohio in the United States were sexually interfertile. Four group 1 strains were not interfertile among themselves or with seven group 2 strains. Attempts to cross G. zeae with representatives of F. acuminatum, F. avenaceum, F. culmorum, F. crookwellense, F. oxysporum, and three mating populations of G. fujikuroi were not successful.     

Breeding for Highly Fertile Isolates of Nectria haematococca MPVI that are Highly Virulent on Pea and In Planta Selection for Virulent Recombinants

ABSTRACT The heterothallic ascomycete Nectria haematococca mating population VI (anamorph Fusarium solani) is a broad host range pathogen. Field isolates of this fungus that are pathogenic on pea tend to be female sterile, of low fertility, and the same mating type (MAT-1), whereas female fertile isolates of either mating type that are highly fertile tend to be nonpathogenic on this plant. To facilitate genetic analysis of traits that may be important in the ability of N. haematococca to parasitize peas, a breeding project was undertaken to produce hermaphroditic isolates of each mating type that are highly fertile and highly virulent on peas. Although the association of high virulence on peas with female sterility and the MAT-1 mating type was not completely broken, isolates with high fertility and high virulence on peas were bred within two generations. Highly virulent progeny were also isolated by an alternative method in which pea plants were inoculated with a mixture of ascospores from a cross between two moderately virulent parents. Whereas all ascospores isolated without selection in planta had lower virulence than the parents, many isolates recovered from diseased tissue were more virulent than the parental isolates. Some of the recovered isolates were shown by restriction fragment length polymorphism analysis to be genetic recombinants of the parents, demonstrating that the pea tissue selected virulent recombinants. All highly virulent isolates tested had the ability to detoxify the pea phytoalexin pisatin, again showing a link between this trait and pathogenicity on the pea.     

Trichothecene profiling and population genetic analysis of Gibberella zeae from barley in North Dakota and Minnesota

Gibberella zeae, the principal cause of Fusarium head blight (FHB) of barley, contaminates grains with several mycotoxins, which creates a serious problem for the malting barley industry in the United States, China, and Europe. However, limited studies have been conducted on the trichothecene profiles and population genetic structure of G. zeae isolates collected from barley in the United States. Trichothecene biosynthesis gene (TRI)-based polymerase chain reaction (PCR) assays and 10 variable number tandem repeat (VNTR) markers were used to determine the genetic diversity and compare the trichothecene profiles of an older population (n = 115 isolates) of G. zeae collected in 1997 to 2000 with a newer population (n = 147 isolates) collected in 2008. Samples were from across the major barley-growing regions in North Dakota and Minnesota. The results of TRI-based PCR assays were further validated using a subset of 32 and 28 isolates of G. zeae by sequence analysis and gas chromatography, respectively. TRI-based PCR assays revealed that all the G. zeae isolates in both populations had markers for deoxynivalenol (DON), and the frequencies of isolates with a 3-acetyldeoxynivalenol (3-ADON) marker in the newer population were ≈11-fold higher than those among isolates in the older population. G. zeae populations from barley in the Midwest of the United States showed no spatial structure, and all the isolates were solidly in clade 7 of G. zeae, which is quite different from other barley-growing areas of world, where multiple species of G. zeae are commonly found in close proximity and display spatial structure. VNTR analysis showed high gene diversity (H = 0.82 to 0.83) and genotypic diversity but low linkage disequilibrium (LD = 0.02 to 0.07) in both populations. Low genetic differentiation (F(ST) = 0.013) and high gene flow (Nm = 36.84) was observed between the two populations and among subpopulations within the same population (Nm = 12.77 to 29.97), suggesting that temporal and spatial variations had little influence on population differentiation in the Upper Midwest. Similarly, low F(ST) (0.02) was observed between 3-ADON and 15-acetyldeoxynivalenol populations, indicating minor influence of the chemotype of G. zeae isolates on population subdivision, although there was a rapid increase in the frequencies of isolates with the 3-ADON marker in the Upper Midwest between the older collection made in 1997 to 2000 and the newer collection made in 2008. This study provides information to barley-breeding programs for their selection of isolates of G. zeae for evaluating barley genotypes for resistance to FHB and DON accumulation.     

Vegetative compatibility groups and parasexual segregation in Colletotrichum acutatum isolates infecting different hosts

Heterokaryosis is an important mechanism which provides genetic variability increase in filamentous fungi. In order to assess the diversity of vegetative compatibility reactions existing among Colletotrichum acutatum isolates derived from different hosts, complementary nit mutants of each isolate were obtained and paired in all possible combinations. Vegetative compatibility groups (VCG) were identified among the isolates according to their ability to form viable heterokaryons. Seven VCGs were identified among the isolates, one of which contained isolates from different hosts. VCGs 2 and 6 contained two and three members, respectively; VCG-3 contained four members, and four VCGs (1, 4, 5, and 7) contained a single one. This study shows, for the first time, the isolation and the parasexual segregation of a heterozygous diploid sector derived from the heterokaryon formed with nit mutants from VCG-6. Diploid, named DE-3, showed nit+ phenotype and growth rate similar to the parental wild isolate. When inoculated in the presence of the haploidizing agent benomyl, the diploid strain produced parasexual haploid segregants exhibiting the nit phenotypes of the crossed mutants. Since viable heterokaryons and diploid may be formed among vegetative compatible isolates of C. acutatum, this study suggests that the parasexual cycle may be an alternative source of genetic variability in C. acutatum isolates.     

灰葡萄孢菌nit突变体的诱导及其在营养体亲和性测定中的应用初探

 从山西运城、临汾、长治、晋中、大同等地保护地黄瓜灰霉病病株上采集、分离的分属于3个不同菌丝融合群的8个灰葡萄孢菌单孢菌株,经氯酸盐诱导处理,共获得了抗氯酸盐的硝酸盐利用缺陷突变体(nit突变体)59株,其中nit1型38株,nit3型10株,nitM型11株。所有nit突变株分别在PDA斜面转管培养3次(21 d)后,除6株恢复成野生菌株外,其余多数nit突变菌株表现稳定。来源于同一野生菌株的不同类型nit突变体间或同一菌丝融合群不同野生菌株的nit突变体间可产生互补反应而形成异核体,其中以nitM型突变株互补性最好,在利用nit突变体测定灰葡萄孢菌营养体亲和性时应作为标准菌株。来源于不同菌丝融合群的nit突变体间不能产生互补反应。     

Use of nitrate non-utilising (Nit) mutants to determine vegetative compatibility in Botryotinia fuckeliana (Botrytis cinerea)

Nitrate non-utilising (Nit) mutants of six field strains and three single-ascospore strains of Botryotinia fuckeliana were selected by plating mycelial plugs onto a nitrate-containing minimal medium amended with chlorate. Mutants were characterised by growth responses on minimal medium amended with various sole nitrogen sources. For each parental strain two mutants were produced: nit1, defective in nitrate reductase apoenzyme; and NitM, defective in the molybdenum cofactor pathway. Complementation on nitrate minimal medium was observed between nit1 and NitM mutants from the same parent in all instances. However, complementation was not observed between any such combinations when the mutants were derived from different parents. It is concluded that a vegetative incompatibility system operates in B. fuckeliana resulting in multiple vegetative compatibility groups. One of the single-ascospore strains was derived from a cross between two of the field strains, indicating that new compatibility groups can be generated during sexual reproduction. Mycelial interaction zones were formed between all parental strains when they were paired on NaCl-amended medium, indicating congruence between mycelial incompatibility and vegetative incompatibility.     

Diversity of Plum pox virus isolates in Bosnia and Herzegovina

Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K₁ with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K₁ typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period.