Effects of indole-3-acetic acid on Botrytis cinerea isolates obtained from potted plants

We study the growth of different isolates of Botrytis cinerea collected from potted plants which were affected by Botrytis blight in southern Spain during recent years. These isolates, which show widely phenotypic differences when grown in vitro, are differentially affected by growth temperature, gibberellic acid applications and paclobutrazol, an efficient plant growth retardant and fungicide at the same time. In this work, we have evaluated the effect of the auxin indole-3-acetic acid (IAA) dose (0, 1, 10, and 100 mg/plate) on the growth of the collection of B. cinerea isolates obtained from the following potted plants: Cyclamen persicum, Hydrangea macrophylla, Lantona camara, and Lonicera japonica. B. cinerea produces indolacetic acid, but so far the precise biosynthetic pathway and some effects on this fungal species are still unclear, although recent studies have revealed an antifungal activity of IAA on several fungi, including B. cinerea isolated from harvested fruits. Mycelial growth curves and growth rates assessed from difference in colony areas during the both linear and deceleration phase, conidiation (measured as time of appearance), conidia length (microm), and sclerotia production (number/plate) were evaluated in the isolates, which were grown at 26 degrees C on Petri dishes containing potato dextrose agar for up to 35 days. Mycelial growth curves fitted a typical kinetic equation of fungi grown on solid media. B. cinerea isolates showed a high degree of variability in their growth kinetics, depending on the isolate and auxin dose. This plant growth substance delayed mycelial growth during the linear phase in an isolate-dependent manner, thus isolates from C. persicum, H. macrophylla and L. camara were more affected by IAA than L. japonica. On the other hand, 100 mg of IAA was the critical dose to significantly reduce the growth rate in all isolates and to promote brown-striped hyphae development, especially in isolate from C. persicum. 10 and 100 mg IAA delayed conidiation in isolates from H. macrophylla but scarcely effects were found in the conidia length. The sclerotia production process was blocked at IAA doses of 100 mg in isolates from L. camara and L. japonica, and was reduced in isolate from H. macrophylla. However, dose of 100 mg IAA had no effect on sclerotia production in isolate from C. persicum. It was concluded that the effect of IAA on B. cinerea growth depends on the isolate, thus isolates from H. macrophylla and L. camara were the most affected by IAA. B. cinerea reduced its development under IAA applications, depending on the isolate and dose. These results confirm those recently published on the inhibitory effect of IAA on Botrytris species growth.     

Benomyl tolerance in isolates of Botrytis cinerea from tomato plants

Three hundred and forty-nine isolates of Botrytis cinerea were collected from tomato crops on forty-one nurseries and 173 (40/6 %) were found to be tolerant to benomyl. There was no obvious association between disease incidence and the occurrence of tolerance. In a fungicide comparison experiment on tomatoes in 1973, twenty of the sixty-four (31 %) isolates examined were benomyl tolerant, the majority of these were from benomyl sprayed plants. In 1974 in a similar experiment, 384 of the 394 (97-5 %) isolates examined were tolerant. Tolerance was monitored in two tomato experiments in relation to a spray programme in which benomyl and dichlofluanid were used in various combinations. There was no marked effect of the spray programmes on the incidence of tolerance on either site. In the experiments B. cinerea was controlled and significant increases in yield were obtained with benomyl in 1973 but not in 1974. This difference is attributed to the change in the pathogen population with a large increase in the incidence of tolerance on the experimental site in 1974.     

Mycoviruses of Botrytis cinerea isolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide‐resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty‐five percent of the B. cinerea isolates analysed contained double‐stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.     

Genetic characterization of grapevine-infecting Botrytis cinerea isolates from Argentina

BackgroundBotrytis cinerea is an ascomycete with a high genetic diversity and complex population structure, as reported from several hosts and sites. However, nothing is known about its genetic diversity in Argentina.AimsThe aim of this work is to estimate the genetic diversity of a local population of B. cinerea isolates obtained from grapevine in Argentina.MethodsIn this work, 35 strains that had been isolated from grapevines were genotyped for the presence of transposable elements and PCR-based RFLP molecular markers. The obtained results were compared with those from a large French population of the fungus, and used to perform a population genetics analysis using the Genepop software.ResultsAll the analysed isolates were classified as Group II (according to the most recent proposed classification) and showed a high degree of genetic diversity, with 14 different haplotypes. A significant difference in allele frequency was recorded between the local and French populations.ConclusionsThese comparisons between fungal populations, led to the detection of a high level of diversity and the differentiation between local and French groups of isolates. This was confirmed by an Fst value of 0.3332, which was higher than that reported for other pairwise comparisons of populations. This work constitutes the first report on the genetic diversity of B. cinerea isolates and their population structure in Argentina.     

源自不同寄主的灰葡萄孢生物学特性的比较研究

本研究以分离自番茄、辣椒、草莓、葡萄的灰葡萄孢Botrytis cinerea为供试菌株,从生长温度、pH适应性、碳源、氮源营养利用等方面对不同寄主来源的灰葡萄孢菌株的生物学性状进行了比较研究。结果表明,5个不同寄主来源的灰葡萄孢菌株的菌丝生长温度范围相同,均为0-35℃;但它们的最适生长温度和分生孢子致死温度存在差异,来自和县番茄菌株HX12最适生长温度为20℃,分生孢子致死温度为47℃ 10min,其余最适生长温度均为25℃,分生孢子致死温度均为48℃ 10min;不同菌株在相同温度下的生长速率有显著差异。pH对不同寄主来源灰葡萄孢菌株菌丝生长的影响存在差异,来自长丰辣椒的菌株LJ菌丝在pH2-9的范围内均能生长,以在pH3-6.5时生长较快,pH6时最快;其余4个菌株在pH2-12的范围内均能生长,以在pH3-9时生长较快,pH6左右最快。不同碳源、氮源营养对灰葡萄孢菌株菌丝生长和分生孢子产生均有显著影响,不同寄主来源的菌株间在碳源、氮源营养利用差异均极显著。在相同碳源、氮源营养条件下,不同寄主来源的菌株的线性生长、菌丝干重和分生孢子产量均有显著差异。     

Comparison of Botrytis cinerea populations isolated from two open-field cultivated host plants

The necrotrophic fungus Botrytis cinerea is reported to infect more than 220 host plants worldwide. In phylogenetical–taxonomical terms, the pathogen is considered a complex of two cryptic species, group I and group II. We sampled populations of B. cinerea on sympatric strawberry and raspberry cultivars in the North-East of Hungary for three years during flowering and the harvest period. Four hundred and ninety group II B. cinerea isolates were analyzed for the current study. Three different data sets were generated: (i) PCR-RFLP patterns of the ADP-ATP translocase and nitrate reductase genes, (ii) MSB1 minisatellite sequence data, and (iii) the fragment sizes of five microsatellite loci. The structures of the different populations were similar as indicated by Nei's gene diversity and haplotype diversity. The F statistics (F_st, G_st), and the gene flow indicated ongoing differentiation within sympatric populations. The population genetic parameters were influenced by polymorphisms within the three data sets as assessed using Bayesian algorithms. Data Mining analysis pointed towards the five microsatellite loci as the most defining markers to study differentiation in the 490 isolates. The results suggest the occurrence of host-specific, sympatric divergence of generalist phytoparasites in perennial hosts.     

Analyses of genetic and pathogenic variability among Botrytis cinerea isolates

Seventy nine isolates of Botrytis cinerea were collected from different host plants and different locations of India and Nepal. All the isolates were identified as B. cinerea based on morphological features and were confirmed using B. cinerea specific primers. Differentiation among the isolates was assessed using morphological, genetic and biochemical approaches. To analyze morphological variability, differences in conidial size, presence or absence of sclerotia and their arrangement were observed. Genetic variability was characterized using RAPD analysis, presence or absence of transposons and mating type genes. Cluster analysis based on RAPD markers was used for defining groups on the basis of geographical region and host. The biochemical approach included determining differences in concentration of oxalic acid and activity of lytic enzymes. All the isolates were categorized into different pathogenic groups on the basis their variable reaction towards chickpea plants. Isolates with higher concentration of oxalic acid and greater activity of lytic enzymes were generally more pathogenic. Pathogenicity was also correlated to transposons. Isolates containing transposa group showed some degree of correlation with pathogenic behavior. However, isolates could not be grouped on the basis of a single approach which provides evidence of their wide diversity and high evolution potential. Sensitivity of sampled isolates was also tested against five botryticides. Most of the isolates from same region were inhibited by a particular fungicide. This feature provided interesting cues and would assist in devising novel and more effective measures for managing the disease.     

Modification of hibiscus growth by treating unrooted cuttings and potted plants with uniconazole or paclobutrazol

Unrooted cuttings ofHibiscus rosasinensis L. “Seminole Pink” were soaked for 5 s in a solution containing 25, 50, 75, or 100 mg L^?1 uniconazole or paclobutrazol, rooted, and then potted and allowed to grow without pinching. Uniconazole was more effective than paclobutrazol in suppressing stem growth and number and length of lateral shoots. Uniconazole and paclobutrazol, at the 25 mg L^?1 concentration, resulted in stem growth 75 and 25%, respectively, of the control, with further reduction at higher rates. Flowering was delayed by the highest rate of uniconazole but not paclobutrazol. Flower number was reduced by both retardants, without any effect on flower size. Plants treated with uniconazole had short pedicels regardless of the rates, whereas paclobutrazol did not affect pedicel length. A second experiment used unrooted cuttings being soaked in a solution containing 0, 12.5, 25, or 50 mg L^?1 uniconazole or having the lower 2.5 cm of the stem dipped in a solution containing 0, 50, 100, or 200 mg L^?1 uniconazole. Plants were pinched after potting. Soaking resulted in more efficient height control than dipping. Lateral shoot number was reduced by soaking but not dipping. All treated plants had smaller stem diameters. Flower size was unaffected regardless of method of treatment and the type of retardant applied. In a third experiment, soil drenches with uniconazole at a rate as low as 0.05 mg/pot resulted in excessive growth retardation. Soil drenches with paclobutrazol at 0.05–0.20 mg/pot reduced shoot growth, flower number, and pedicel length, but did not affect days to bloom.     

Modification of hibiscus growth by treating unrooted cuttings and potted plants with uniconazole or paclobutrazol

Unrooted cuttings ofHibiscus rosasinensis L. Seminole Pink were soaked for 5 s in a solution containing 25, 50, 75, or 100 mg L^–1 uniconazole or paclobutrazol, rooted, and then potted and allowed to grow without pinching. Uniconazole was more effective than paclobutrazol in suppressing stem growth and number and length of lateral shoots. Uniconazole and paclobutrazol, at the 25 mg L^–1 concentration, resulted in stem growth 75 and 25%, respectively, of the control, with further reduction at higher rates. Flowering was delayed by the highest rate of uniconazole but not paclobutrazol. Flower number was reduced by both retardants, without any effect on flower size. Plants treated with uniconazole had short pedicels regardless of the rates, whereas paclobutrazol did not affect pedicel length. A second experiment used unrooted cuttings being soaked in a solution containing 0, 12.5, 25, or 50 mg L^–1 uniconazole or having the lower 2.5 cm of the stem dipped in a solution containing 0, 50, 100, or 200 mg L^–1 uniconazole. Plants were pinched after potting. Soaking resulted in more efficient height control than dipping. Lateral shoot number was reduced by soaking but not dipping. All treated plants had smaller stem diameters. Flower size was unaffected regardless of method of treatment and the type of retardant applied. In a third experiment, soil drenches with uniconazole at a rate as low as 0.05 mg/pot resulted in excessive growth retardation. Soil drenches with paclobutrazol at 0.05–0.20 mg/pot reduced shoot growth, flower number, and pedicel length, but did not affect days to bloom.     

灰葡萄孢强除草菌株的筛选与活性物质的组分分析

灰葡萄孢产生的毒素具有较强的除草活性,开发成生物除草剂前景广阔。本试验以灰葡萄孢BC4为出发菌株,通过紫外线和化学诱变剂诱变分生孢子或菌丝体,获得了所产毒素除草活性比野生菌株显著增强的3个突变菌株。对各菌株产生的毒素提取液进行HPLC分析,发现获得的3个突变菌株中各个组分的含量均显著高于野生菌株,且遗传稳定。利用HPLC制备得到了4个组分,其中组分Ⅰ、Ⅳ对马唐和反枝苋具有很强的杀除活性,为灰葡萄孢的遗传改良及毒素源除草剂的工业化生产奠定了基础。     

Effects of fungicide spray regimes on incidence of dicarboximide resistance in grey mould (Botrytis cinerea) on strawberry plants

In tunnel experiments, the efficacy of dicarboximide sprays in controlling grey mould of strawberries was greatly decreased by the presence of dicarboximide-resistant forms of Botrytis cinerea. The use of dichlofluanid, as a tank-mix or in an alternating programme, with a dicarboximide fungicide, procymidone, helped to maintain the efficacy of disease control but failed to prevent an increase in the proportion of dicarboximide-resistant forms of the pathogen. Alternative ‘partner’ fungicides (thiram, chlorothalonil) delayed build-up of resistance to dicarboximides. Build-up of resistance was absent or relatively small in unsprayed plots. Application of dichlofluanid alone was always associated with a substantial increase in dicarboximide resistance, although less than in procymidone-treated plots. Monitoring dicarboximide resistance in the tunnels during the winter, when no further sprays were applied, revealed a gradual decline in the proportion of dicarboximide-resistant forms in all previously treated plots. In laboratory studies on inoculated leaf debris, dichlofluanid treatment induced the build-up of dicarboximide-resistant forms of B. cinerea. Leaf-disc tests revealed cross-resistance of dicarboximide-resistant isolates towards dichlofluanid but not towards thiram or chlorothalonil. Dichlofluanid is widely used for control of B. cinerea and the implications of these results for the practical management of dicarboximide resistance in this pathogen are discussed.     

多效唑和矮壮素对盆栽彩色马蹄莲的矮化实验

采用200～400 mg*L-1多效唑(PP333)和1 000～2 000 mg*L-1矮壮素(CCC)对处于生长中期的2个彩色马蹄莲(Zantedeschia antedeschia)盆栽品种进行矮化处理,结果显示彩色马蹄莲的株型明显矮化,茎杆增粗.统计分析表明:PP333处理的矮化效果较明显,其中以300 mg*L-1效果最佳,说明PP333对设施栽培条件下彩色马蹄莲的防徒长、抗倒伏的矮化处理有实用价值.     

番茄灰霉病拮抗内生放线菌的筛选、鉴定及其活性评价

对安徽省淮北市番茄植株根、茎、叶中内生放线菌进行了分离、筛选,并测定了其抑菌活性。结果表明:番茄根、茎和叶中的内生放线菌的数量分别为5.66×104、0.67×104和0.39×104CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到93株内生放线菌,通过对峙实验,筛选到7株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生放线菌总数的7.5%。来自根组织中的菌株HNU-EA27的抑菌效果最佳,抑菌圈直径达28.3mm。根据形态特征、培养特征、生理生化特性、细胞壁组分和16SrDNA序列分析,将菌株HNU-EA27鉴定为毒三素链霉菌(Streptomyces toxytricini)。室内测定菌株HNU-EA27发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明:菌株HNU-EA27发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为30%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明:30%菌株HNU-EA27发酵滤液对番茄灰霉病的预防与治疗效果分别为80.6%和73.8%,均高于50%多菌灵可湿性粉剂600倍液。本研究表明,菌株HNU-EA27是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

A new double-stranded RNA mycovirus from Botrytis cinerea

A simple double-stranded RNA mycovirus was detected in a wild-type Botrytis cinerea 55k strain. The virus was located in the fungus cytoplasm as free particles of approximately 28 nm in diameter. The mycovirus possesses a single double-stranded genome segment of 1.8 kilobase pairs (kbp) encapsidated within an isometric protein coat whose main structural component is a polypeptide of 68 kDa. Cells infected with this virus showed an important degree of cellular degeneration.     

灰葡萄孢BC7-3菌株除草活性组分的纯化与结构鉴定

[目的]植物病原真菌毒素是一类重要的微生物源除草剂,本研究旨在找到一个新的具有除草活性的化合物结构.[方法]在前期薄层层析法、柱层析法和高效液相色谱法分析的基础上对灰葡萄孢诱变菌株BC7-3的代谢产物中具有除草活性的5个不同组分分别进行了液相色谱制备.[结果]本研究得到了一个纯度达99.38%对单子叶杂草马唐具有较强杀除活性的纯组分,通过对纯组分的物理性状测定并结合紫外-可见吸收光谱、红外光谱以及核磁共振波谱等分析方法鉴定化学结构为10-顺-二氢化灰霉二醛.[结论]研究的结果为微生物除草剂的创新和开发奠定了理论基础.     

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.     

番茄几种挥发性组分对番茄灰葡萄孢的抑制作用

采用孢子悬滴培养法,测定番茄几种挥发性组分对番茄灰葡萄孢(Botrytiscinerea)孢子萌发及菌丝生长的抑制作用。结果表明各挥发性组分对灰葡萄孢均有抑制作用,抑制率的大小依次为挥发醛、芳香族化合物和萜类化合物。10μmol/L的(E)-2-己烯醛和壬烯醛可完全抑制孢子萌发和菌丝生长,4μmol/L的丁子香酚和10~20μmol/L的水杨酸甲酯使病菌菌丝异常生长。本研究为番茄挥发性物质在其抗病防御反应中的作用提供理论依据。