影响活体内马铃薯晚疫病菌卵孢子产生因素的研究

研究了不同菌株组合,马铃薯植株茎、叶及接种物中A1和A2菌株孢子囊比例、温度、湿度对卵孢子产生的影响。不同菌株组合产生卵孢子的数量有显著差异;在离体接种情况下,叶片中产生卵孢子数量大于茎中产生卵孢子数量;A1和A2菌株中孢子囊不同比例对卵孢子产生影响很大,当比例为1：1时卵孢子产生量最大;15℃光照条件下培养,并给侵染叶片持续的水分供应才能产生大量卵孢子;寄主的抗性水平对卵孢子产生有明显的影响,中抗品种上产生卵孢子量最多,高抗品种上产生卵孢子量最少,感病品种上产生卵孢子量居中。     

马铃薯晚疫病菌卵孢子萌发的初步研究

描述了马铃薯晚疫病菌卵孢子萌发的方式并研究了菌株组合、卵孢子形成时间、在琼脂培养基上培养时间及光照对卵孢子萌发的影响,结果表明不同菌株组合卵孢子萌发率为0—7.2%,对峙培养20天后形成的卵孢子的萌发率最高达8.7%,在琼脂培养基上培养25-30d 萌发率最高达11.4%,卵孢子形成时黑暗及在琼脂培养基上萌发时光照萌发率最高达11.8%。     

致病疫霉在中国云南的马铃薯田间形成卵孢子*

由致病疫霉Phytophthora infestans (Mont.)de Bary引起的马铃薯和番茄晚疫病是世界性的作物病害,每年均造成巨大的经济损失和社会影响。致病疫霉是异宗配合的卵菌,有两个已知交配型A1和A2,两个相对交配型互作时可进行有性生殖产生卵孢子( Gallegly & Galindo,1958)。过去许多年一直认为致病疫霉在除墨西哥以外的国家中只存在A1交配型,通过产生孢子囊进行无性繁殖。近年来,由于致病疫霉新致病群体的产生以及全球范围的迁移和替代,A2交配型菌株先后在欧洲、美洲、亚洲和非洲的许多国家被发现。A1、A2两种交配型菌株的同时存在,增…     

培养条件对马铃薯晚疫病菌卵孢子产生的影响*

马铃薯晚疫病菌（Ｐｈｖｔｏｐｈｔｈｏｒａ　ｉｎｆｅｓｔａｎｓ）在我国能够进行有性生殖产生卵孢子^［１］,研究了离体培养时培养基成份、温度、光照及ｐＨ值对卵孢子产生的影响。结果表明：白云豆培养基、小米培养基和大豆培养基均适合卵孢子的产生,产生的数量为１３１．６～１４９．６个／ｃｍ^２,但冰冻豌豆培养基不适合卵孢子的产生,产生的数量仅为５．８个／ｃｍ^２。比较不同ｐＨ值、不同温度、不同光照条件对卵孢子产生的影响发现,ｐＨ值为７、温度为１     

培养条件对马铃薯晚晚疫病菌卵孢子产生的影响

马铃薯晚疫病菌（Ｐｈｙｔｏｐｈｔｈｏｒａ ｉｎｆｅｓｔａｎｓ）在我国能够进行有性生殖产生卵孢子＾「１」,研究了离体培养时培养基成份、温度、光照及ｐＨ值对卵孢子产生的影响。结果表明：白云豆培养基、小米培养基和大豆培养基均适合卵孢子的产生,产生的数量为１３１．６～１４９．６个／ｃｍ＾２,但冰冻豌豆培养基不适合卵孢子的产生,产生的数量仅为５．８个／ｃｍ＾２。比较不同ｐＨ值、不同温度、不同光照条件对卵孢子     

马铃薯晚疫病菌DK98-1、DX98-2和DX98-3的生物学特性及AFLP指纹分析

利用黑麦培养基和V8-蔬菜汁培养基研究了马铃薯晚疫病菌Phytophthora infestans特异菌株DK98-1、DX98-2和DX98-3的生物学特性,发现该菌株与普通菌株相比菌落生长速度慢、孢子囊产生数量少、有性杂交后卵孢子产生量大(2047～75623个/cm2);利用AFLP分子标记研究这3个菌株的DNA指纹图谱,发现用引物E CG/M CC扩增菌株DK98-1、DX98-2和DX98-3后,在330bp处与普通菌株相比各缺失一条谱带,用引物E AC/M CT扩增菌株DK98-1、DX98-2和DX98-3后,在370bp处比普通菌株增加1条谱带,说明这3个菌株与普通菌株在遗传上明显不同。同时可以利用上述2对特异性引物,鉴定在自然界的晚疫病菌群体中这类特异菌株的出现频率。     

马铃薯晚疫病菌DK98-1、DX98-2和DX98-3的生物学特性及AFLP指纹分析

利用黑麦培养基和V8-蔬菜汁培养基研究了马铃薯晚疫病菌Phytophthora infestans特异菌株DK98-1、DX98-2和DX98-3 的生物学特性,发现该菌株与普通菌株相比菌落生长速度慢、孢子囊产生数量少、有性杂交后卵孢子产生量大（2047～75623个/cm²）;利用AFLP分子标记研究这3个菌株的DNA指纹图谱,发现用引物E+CG/M+CC扩增菌株DK98-1、DX98-2和DX98-3后,在330bp处与普通菌株相比各缺失一条谱带,用引物E+AC/M+CT扩增菌株DK98-1、DX98-2和DX98-3后,在370bp处比普通菌株增加1条谱带,说明这3个菌株与普通菌株在遗传上明显不同。同时可以利用上述2对特异性引物,鉴定在自然界的晚疫病菌群体中这类特异菌株的出现频率。     

进境美国加州脐橙中丁香疫霉Phytophthora syringae的截获

从产自美国加利福尼亚州的新鲜脐橙样品中发现多个腐烂病果,通过分离培养得到3个疑似丁香疫霉Phytophthora syringae菌株,对3个菌株进行形态学研究、致病性测定和分子序列比对分析。结果表明病菌在V8A培养基上菌落稀疏、平铺,呈星状,菌丝紧贴培养基生长或埋于基质内生长;在PDA培养基上菌落呈菊花花瓣状,菌丝致密,乳白色;游动孢子囊和菌丝膨大体在无菌水和土壤浸出液中黑暗条件下48h后产生;菌株为同宗配合,卵孢子在带有新鲜脐橙果实组织或杜鹃叶片的V8A培养基中大量产生;创伤接种脐橙果实,7d后接种脐橙出现典型的褐腐症状;通用引物ITS1/ITS4扩增测序,Blastn分析表明序列与GenBank中P. syringae序列相似性为99%。依据上述研究结果,将分离获得的3株菌鉴定为丁香疫霉Phytophthora syringae,系国内首次截获的一种植物检疫性真菌病害。     

莴苣叶疫病和茎腐病的病原

2021年3-6月,从甘肃省的生菜(叶用莴苣)叶片和青海省的莴笋(茎用莴苣)茎秆罹病样本上分离得到腐霉属卵菌。通过Koch’s法则明确了分出菌株的致病性。依据形态学和分子生物学特征,将3个菌株鉴定为嗜导管腐霉Pythium tracheiphilum。在核糖体DNA内转录间隔区(rDNA-ITS)、细胞色素氧化酶亚基1 (cox 1)和核糖体DNA 28S大亚基(rDNA-LSU)基因联合系统发育树中,甘肃菌株(LPy-B)和青海菌株(LPy-C和LPy-D)被聚在P. tracheiphilum的不同亚群里,不同菌株的适宜生长温度和产孢特性存在差异。孢子囊顶生、间生或侧生,球形,17.13-53.73 μm,或近球形至葫芦状,24.58-56.72×18.62-53.73 μm;休止孢球形,6.70-9.68 μm;藏卵器光滑,顶生或间生,球形,15.64-23.09 μm;每个藏卵器有雄器1-2个,雄器与藏卵器同丝或异丝生;卵孢子满器或近满器,球形,直径13.41-20.11 μm,卵孢子壁厚0.74-2.23 μm。致病性测定结果表明,除莴苣外,嗜导管腐霉还可侵染菊科的华蒲公英和刺儿菜、十字花科的青菜、豆科的蚕豆;不侵染车前科的车前、马齿苋科的马齿苋和苋科的反枝苋;在试验条件下,菌株LPy-C和LPy-D对青菜叶片的致病性强于LPy-B。嗜导管腐霉及其引起的莴苣叶疫和茎腐病在我国和亚洲地区系首次报道。     

掘氏疫霉遗传学研究II．交配型遗传与变异的研究

本研究以掘氏疫霉P“yl口外埔o,o dr~cksleri丁ucke r野生型菌株的单游动孢子无性系为亲本,测定了自交、杂交后代交配型的遗传,经Kmno·处理引起的交配型变异以及Ft代可自孕单卵孢株有性生殖后代交配型的遗传与变异。聚碳膜间隔配对诱导A,和^,菌株自交产生的卵孢子经H zot处理索0激萌发(萌发率1 z～16％)获得单卵孢株。交配型测定结果表明,^t和A,亲本自交st代单卵孢株均保持与亲本一致的交配型。用KMnO．处理上述卵孢子导致A-和^z亲本的部分s一代单卵孢株出现自孕现象,少数A,亲本自交后代改变为A：交配型;从A。交配型亲本的s-代可自孕菌株产生的卵孢子萌发所建立的单卵孢株中,同时获得扎和^。交配型菌株。上述结果不支持San somc关于疫霉菌AhA,交配型分别由纯合、杂合基因控制的假说,进一步证明了Ko关于交配型抑制因子控制交配型表达的假说的合理性。 掘氏疫霉种内菌株直接配对产生的卵孢子用HtO,刺激萌发获得Ft代单卵孢拣。测定结果表明,在F。代出现A。A,、A,A;、A口4种交配型的单卵孢株,其比例因不同亲本组合而有较大差异oF。代出现的^。A,菌株自孕产生的卵孢子经HtO,处理刺激萌发建立自交系,观察到^。^：交配型在有性生殖后代发生分离,各交配型比例因亲本不同而异o A·At菌株的自孕能力在单游动孢子无性系后代可稳定遗传,认为F-代出现的AJAt个体来自亲本的杂交。AtA-单卵孢株保藏4～6个月后大多仍具自孕能力,少数改变为Az交配型。     

Oospore Germination and Formation by the Late Blight Pathogen Phytophthora infestans in vitro and under Field Conditions

The ability of the late blight pathogen Phytophthora infestans to form oospores in leaves of seven potato cultivars was examined at different incubation temperatures under controlled environmental conditions and under field conditions. At 10°C, the oospore formation in three intermediate-resistant cultivars all differed significantly from each other (P < 0.05), with the lowest amount formed in cv. Asterix. This latter cultivar did not form oospores at any other temperature. Under field conditions oospores were formed abundantly in a naturally infected field. A significant date by cultivar interaction showed that P. infestans increased the oospore formation in foliage by time in cvs Columbo, Hertha and Matilda, whereas no significant differences between dates were found for other cultivars. The genetic structure of P. infestans in the naturally infected field plot, where oospores formed abundantly, was studied by using amplified fragment length polymorphism and a high genetic diversity was revealed. Oospore germination from two Scandinavian (A1 and A2) P. infestans isolates was stimulated in visible light and in 1 : 2 and 1 : 10 soil extract. The effect of light and nutrients on oosporogenesis is discussed.     

Production of Phytophthora infestans oospores in planta and inoculum potential of in vitro produced oospores under temperate highlands and subtropical plains of India

High moisture content of the host tissue ( 88%) and low ambient r.h. (50-54%) favoured oospore formation under controlled environments. It took 14–16 days for oospores to develop; thereafter the number of oospores increased with time and decreased with moisture content of host tissue. High ambient r.h. (> 80%) did not favour oospore formation under field or controlled conditions. Oospore formation was detected in inoculated plants grown in the field when the ambient r.h. declined to 74% and moisture content of host tissue decreased to 83.7–85.6%. It took 8 days (cv. Kufri Chandramukhi) to 13 days (cv. Kufri Jyoti and Kufri Badshah) for oospores to develop. Cultivars also differed in their response to oospore production, cv. Kufri Chandramukhi being more responsive (4800 oospores g⁻¹ f wt) than cv. Kufri Jyoti and Kufri Badshah (1320 and 390 oospores g⁻¹ f wt respectively). Oospores produced in vitro remained viable when buried in soil in the temperate highlands of Himachal Pradesh and sub-tropical plains of Uttar Pradesh, India for more than 150 days, i.e. beginning of the next crop season. The oospores germinated and initiated late blight infection at the base of the stems after 21–30 days of incubation of the potato plants raised in oospore-infested soil. It took 2 days for newly formed oospores to germinate and this delay time increased to 75–77 days after 180-days burial. It took 15 days for their germination (47%) in soil extract as compared to 50 days in sterilised distilled water.     

Oospores progenies from Phytophthora ramorum

Oospores of Phytophthora ramorum were produced from intraspecific pairings between a European A1 and European or American A2 strains. Their viability was evaluated through colouration with tetrazolium bromide. The distribution of oospores in the different classes of colouration was similar to that found in other Phytophthora species (homothallic and heterothallic): most of the oospores stained purple, which corresponds to spores in dormancy. In order to produce single-oospore cultures, a method was developed to separate oospores from mycelium and chlamydospores. Germination of oospores was observed in 110, 250, 350 and 500-d-old cultures at a low proportion. Microsatellite marker analyses on oospore progenies revealed that the oospores resulted from hybridisation. More than 50 oospore progenies were characterised in terms of mating type, aggressiveness on Rhododendron leaves, and growth rate on two different media. The results are discussed in the context of pest risk analysis.     

Development of a Defined Medium for Pythium oligandrum Oospore Production

Complex and defined media have been previously proposed for production of oospores of Pythium oligandrum , a fungal mycoparasite of several disease-causing fungi. However, oospore production in synthetic media requires long periods of incubation and yields lower oospore numbers than in complex media. Moreover, although complex media produce high oospore yields, these yields are not reproducible because of the variability in the composition of complex nutrient sources. In the present study, the average composition of molasses reported in the literature was utilized as a base to develop a new defined medium for P. oligandrum oospore production. Forty-two substrates defined in nine stock solutions: carbohydrates, vitamins, sterols, non nitrogenous acids, amino acids, minerals, nucleic acid bases, CaCl 2 and MgSO 4 were tested at two levels (present or absent) in two fractional factorial designs. Each of these nine variables had a significant main effect on oospore production. Furthermore, the effect of each variable, except for vitamins, depended on the level of each other variable, expressed by two-variable interactions. The maximal predicted oospore production was calculated from the polynomial regressions associated with the two fractional factorial designs. Oospore productions were 1.3 and 2 ×10 6 oospores mL -1 from the first and second designs. In order to optimize the oospore production, the two levels of each variable were modified and all variables were experimented in a selection of a complete factorial design (Plackett and Burman design). A fitted first-order polynomial regression equation provided the combination of levels of variables for optimal oospore production. A defined medium, based on this combination of levels, was used for P. oligandrum growth. The optimized oospore production after 7 days growth was 4 ×106 oospores mL -1 as predicted by the polynomial regression. Oospore yields, biomass produced from oospores and oospore freeze-drying tolerance were similar when P. oligandrum was previously grown in molasses or in the new defined medium.     

A Comparison of the Resistance Against Phytophthora fragariae var. rubi, the Causal Agent of a Raspberry Root Rot

Zoospore encystment, sporulation, oospore formation and detection of Phytophthora fragariae var. rubi , the causal agent of raspberry root rot, on three Rubus genotypes with different levels of resistance were investigated. There were no qualitative or quantitative differences in encystment as determined by scanning electron and fluorescence microscopy. Susceptibility was associated with an extended period of sporulation on roots with a proportion of roots containing oospores increasing continuously until the whole root system was colonised and the fungus could be isolated from the stem bases. Quantitative resistance as found in some raspberry ( Rubus idaeus ) cultivars was associated with a shorter sporulation period and decreasing proportion of the roots containing oospores due to the strong regeneration of healthy roots and limited spread of the fungus in the root system. Stem bases were consequently not affected. Sporulation was very limited on a blackberry x raspberry hybrid that was almost totally resistant to root rot and sustained infections could not be found.     

Phytophthora kernoviae oospore maturity, germination, and infection

Limited information is known on the basic biology of the recently described Phytophthora kernoviae that produces homothallic oospores. In this study, different P. kernoviae isolates were used to investigate oospore maturity, germination, and infection. All isolates produced oospores in V8 broth at 20°C in the dark by 6d. Oospores also formed at 10 and 15°C, but did not form at 25 and 28°C. Continuous light inhibited oospore production of some isolates but had no negative effect on others. Maturation time of the oospores, as noted by germination and staining with tetrazolium bromide, was not much different among the isolates between 2 and 14 weeks. Oospore germination was optimal at 18 and 20°C, and did not occur at 5, 25, and 30°C. Oospore germination under continuous light was higher than in the dark, but individual isolates showed variable results. Rhododendron leaf disks inoculated with oospores and maintained in the dark at 20°C were necrotic after 1 week, while those kept under continuous light did not develop necrosis. The percentage of leaf disks infected with P. kernoviae was lower in the leaves exposed to continuous light (40%) compared to those kept in the dark (100%).     

Extraction from Plant Tissue and Germination of Oospores of Peronospora viciae f.sp. pisi

A protocol was developed to extract oospores of Peronospora viciae f.sp. pisi from plant tissue and control bacterial contamination in a germination assay. Oospores were extracted by comminuting infected leaves and pods in water, sonicating the suspension and sieving it through mesh sizes 53 and 20 pm, respectively. Germination of oospores was negatively influenced by addition of chloramphenicol and penicillin. A combination of ampicillin and rifampicin strongly inhibited bacterial growth at 10 C, and did not negatively affect germination. Washing oospores in water or 0.02% Tween-80, and sonication did not influence germination. Treating oospore suspensions with cellulase buffered at pH 4.6 for 2 h digested most plant tissue but did not influence germination. Incubation in 0.05 m acetate buffer delayed germination. Germination was unaffected when oospores were incubated in 0.05 m citrate buffer.     

Components of Resistance to Early Blight in Four Potato Cultivars: Effect of Leaf Position

Components of early blight resistance were quantified in leaves of different ages in four potato cultivars. The components of resistance: incubation period (IP), lesion number (LN), early blight severity, lesion expansion rate (LER), latent period (LP) and spore production by lesion area (SPLA), were evaluated separately in the lower, middle and upper leaves of four potato cultivars. Plants of cultivar Aracy (resistant), Delta (moderately resistant), Desirée (susceptible) and Bintje (susceptible) were inoculated with an Alternaria solani isolate at the beginning of the flowering stage. Disease severity varied in different plant parts. In all cultivars, regardless of resistance, the smallest values of LN, and severity were recorded on the upper leaves, suggesting that young tissues are less susceptible. In cultivar Aracy, the IP was long, with small values of LN and LER and consequently, low values of early blight severity in all leaf positions were recorded. Although IP was long in cultivar Aracy, no differences between the moderately resistant cultivar Delta and the susceptible cultivars Bintje and Desirée could be detected for this component. The IP was only influenced by leaf position in cultivar Aracy. Clear differences in resistance levels among cultivars could be detected regarding LN, severity and LER. However, neither LP nor SPLA were associated with resistance level of cultivars or with leaf position. Analyses according to plant part suggest that evaluations on leaves of the middle third part are most suitable for screening for early blight resistance in potato.     

Inhibition of Chara vulgaris oospore germination by sulfidic sediments

The germinability of Chara vulgaris oospores collected from the sediments of four Ontario lakes varies considerably, ranging in germination percentage from 7% to 54%. Chemical analysis of the interstitial water of the sediments indicated that oospores with low germination occurred in lakes which have high acid volatile sulfides (H₂S, FeS, HS⁻) and high soluble Fe²⁺. The inhibitory effects of sediment on oospore germination were demonstrated by transplant experiments, and suggested that sulfide was the toxic agent. Exposure of high-germinating sedimentary oospores to free sulfide concentrations greater than 2.0 mM caused a greater than 30% reduction in oospore germination. The presence of sulfide in sediments was shown to result from sulfate reduction by bacteria in sediment pore water of those lakes where oospore viability was lowest. Differences in oospore germination percentage appear, therefore, to be due to the toxicity of H₂S produced in the sediment, either by a direct effect on the oospore, or on the parent plants.