Antifungal Activity of 2-Deoxy-D-Glucose on Botrytis cinerea, Penicillium expansum, and Rhizopus stolonifer: Ultrastructural and Cytochemical Aspects

ABSTRACT The effect of 2-deoxy-D-glucose on major postharvest pathogens was investigated at the ultrastructural and cytochemical level. Hyphae of Botrytis cinerea, Penicillium expansum,, and Rhizopus stolonifer grown in the absence of 2-deoxy-D-glucose were normal and showed no apparent cytological alterations. In the presence of 2-deoxy-D-glucose, however, these fungi exhibited severe cellular injuries ranging from cell wall disruption to cytoplasm disintegration. Although 2-deoxy-D-glucose caused cytoplasmic degeneration in the three fungi tested, cell wall alterations were exhibited only by B. cinerea and R. stolonifer. In the latter, the retraction of degenerated cytoplasm was often accompanied by the deposition of amorphous material in paramural spaces. Cytochemical study of fungal cell wall components showed that 2-deoxy-D-glucose caused a marked increase of chitin- and beta-1,3-glucan-labeling in R. stolonifer and B. cinerea, indicating an interference of 2-deoxy-D-glucose with fungal wall biosynthesis. The observed cellular alterations indicate that 2-deoxy-D-glucose may also have affected other metabolic processes.     

Effects of the experimental fungicide RH 886 on Mucor piriformis

No registered fungicide controls Mucor piriformis, a cause of severe postharvest storage rot in pears, but the experimental fungicide RH 886 (active ingredients: 77% 5-chloro-2-methylisothiazol-3-(2H)-one and 23% 2-methylisothiazol-3-(2H)-one) has an ED₅₀ of 23.1 μg ml^?1 in 5 min exposure for germination of sporangiospores of M. piriformis and an ED₅₀ of 9.9 μg ml^?1 for mycelial growth. Mixing RH 886 into infested, amended soil at 8 mg g^?1 soil or mixing copper sulfate into soil at 1 mg g^?1 soil prevented sporulation of M. piriformis. Application of RH 886 to pear fruits prior to inoculation, or immersion of fruits in solutions of RH 886 containing sporangiospores of M. piriformis significantly reduced fruit infection.     

Effets de divers herbicides sur la croissance mycélienne de souches de Botrytis cinerea pers et de Penicillium expansum Link, sensibles ou résistantes à certains fongicides

Effects of various herbicides on mycelial growth of strains of Botrytis cinerea. Pers. and Pénicillium expansum Link, sensitive or resistant to certain fungicides Of seventy herbicides tested, thirty-eight were slightly toxic to B. cinerea (CI50, concentration giving 50% inhibition of the speed of mycelial growth, exceeding or equal to 100g of herbicide/ml of nutrient solution) and, of those, ten had no effect even at 1000 g/ml. The most active products (CI50 less than 10 g/ml) were Chlorthiamid, dichlobénil, nitrofen, propyzamide and phenolic derivatives (DNOC, ioxynil, PCP). In most cases P. expansum was less sensitive than B. cinerea except to endothal, propachlor, prynachlor and certain substituted ureas. Strains resistant to the benzimidazole fungicides (carbendazim, thiabendazole etc.) show increased sensitivity to certain carbamade herbicides (barban, chlorbufam etc.), this indicates the existence of a negative cross resistance between these groups of antimitotic pesticides. Strains resistant to the cyclic-imide fungicides (iprodionc, procymidone, vinclozolin) and to various aromatic compounds (biphenyl. chloroneb, dicloran etc.) may also be resistant to bipyridilium, dinitroanilinc and diphenylethcr herbicides, to chlorthamid. dichlobénil and oxadiazon. This cross resistance between pesticides with different biochemical modes of action has yet to be explained.     

Effects of temperature,relative humidity and age of conidia on the incidence of spotting on gerbera flowers caused by Botrytis cinerea

In recent years, spotting of ray florets of gerbera flowers has become an important problem. This type of small necrotic lesions may occur before, but especially shortly after harvesting the flowers.Botrytis cinerea was easily isolated from such lesions. Inoculation withB. cinerea only gave typical necrotic lesions, when dry conidia were dusted on the flowers with a short period of high rh after inoculation. At 18–25 °C a high rh for at least 5 hours was necessary. Rotting of ray florets and receptacles byB. cinerea occurred when inoculated flowers were kept wet for a few days. Spots consist of one to several necrotic, usually epidermal cells. A single conidium could give rise to a necrotic lesion after germination. Germination of conidia and lesion formation occurred between 4 and 25 °C; at 30 °C, germination and lesion formation did not occur. Between 18 and 25 °C, many lesions became visible within 1 day after inoculation; at 4 °C it took 2 to 3 days before lesions could be seen. If kept dry, conidia ofB. cinerea remained ungerminated on ray florets of gerbera flowers and could be removed from the ray florets. Within 1 day at high rh, germination occurred and lesions were produced. Conidia ofB. cinerea, stored dry, were able to survive much longer than the lifetime of a gerbera flower. Even after storage at room temperature for up to 14 months, some conidia were able to germinate in vitro and on ray florets and induce the formation of lesions. Addition of gerbera pollen diffusate stimulated germination and lesion formation.     

Effect of inoculum composition on infection of French bean leaves by conidia of Botrytis cinerea

Inoculation of leaves of French bean (Phaseolus vulgaris) with sprays or small drops of a suspension of conidia ofBotrytis cinerea gave rise to spreading lesions, lesions remaining restricted in size or to no visible necrosis. The type of reaction depended on the composition of the inoculum. In studies with drop inoculations with buffered inocula some of the factors involved were analyzed. The formation of spreading lesions depended on pH, type and molarity of the buffer, presence of glucose, and concentration of conidia in the inoculum. If the phosphate buffer used in most of the inocula was replaced by monobasic phosphate, similar results were obtained. The reactions were not influenced by the proportion of K⁺ or Na⁺ ions in the phosphate buffer. Inoculations with conidia suspended in a solution of 0.067 M phosphate buffer (pH 5.0) or monobasic phosphate and 0.11 M glucose always evoked a susceptible reaction, i. e. the formation of spreading lesions.     

Effects of Ca2+ and Mg2+ on Botrytis cinerea and Penicillium expansum in vitro and on the biocontrol activity of Candida oleophila

Calcium salts have been reported to play an important role in the inhibition of postharvest decay of apples and in enhancing the efficacy of postharvest biocontrol agents. Therefore, the present study was conducted in order to examine and compare the effects of calcium and magnesium salts on the germination and metabolism of the postharvest pathogens Botrytis cinerea and Penicillium expansum , and to determine the effects of these salts on the biocontrol activity of two isolates (182 and 247) of the yeast Candida oleophila. Increasing concentrations of CaCl₂ (25–175 mM) resulted in decreased spore germination and germ-tube growth of both pathogens. The greatest effect was observed in the case of B. cinerea. The inhibitory effect could be overcome by the addition of glucose to the germination medium. MgCl₂ (25–175 mM) had no effect on germination or germ-tube growth of either pathogen, indicating that the calcium cation rather than the chloride anion was responsible for the inhibition. The pectinolytic activity of crude enzyme obtained from the culture medium of both pathogens was also inhibited by 25–175 mM CaCl₂, with the greatest effect on the crude enzyme from P. expansum. Biocontrol activity of isolate 182 was enhanced by the addition of 90 or 180 MM CaCl₂, whereas there was no effect on the biocontrol activity of isolate 247. This was apparently due to the inability of isolate 247 to proliferate in apple wounds. It is postulated that enhanced biocontrol activity of isolate 182 of the yeast C. oleophila in the presence of Ca²⁺ ions is directly due to the inhibitory effects of calcium ions on pathogen spore germination and metabolism, and indirectly due to the ability of isolate 182 to maintain normal metabolism in the presence of"toxic" levels of calcium.     

Horizontal and vertical distribution of airborne conidia of Botrytis cinerea in a gerbera crop grown under glass

The horizontal and vertical distribution of airborne conidia ofBotrytis cinerea in a gerbera crop in two glasshouses (100 m² and 350 m²) was studied during 18 months in 1988 and 1989. Conidia ofB. cinerea were caught in simple spore traps consisting of agar in Petri dishes placed in a regular pattern at three different heights in the glasshouse and counted as colonies, after incubation. Lesions due to conidial infection were counted on gerbera petals. The horizontal and vertical distribution of conidia ofB. cinerea in a gerbera crop grown under glass was fairly uniform in both distinct glass-houses. Conidia ofB. cinerea trapped in a glasshouse can originate from sources inside and outside the glasshouse. No significant interaction was found between location and time for the colony counts and for the log transformed (ln(N+1)) lesion counts. The results of this study suggest that spore trapping at one height and at a limited number of locations and dates is sufficient for efficient monitoring ofB. cinerea in a glasshouse.     

温度调控对番茄灰霉病菌侵染的影响

从群体的角度研究了番茄灰霉病菌对温度的适应性,结果表明灰霉病菌孢子的萌发适温在15～25℃,最适温度为20℃。15～25℃适宜菌丝生长、侵染与扩展,20℃为最适温度。30℃以上病害发展受到抑制。低温有利于病菌产孢,最适温度为15℃。变温研究表明,在一天中,随着30℃以上高温时间的增长,病菌(菌丝)的致病力呈下降趋势,6 h以上高温能够限制病害发展。     

湿度调控对番茄灰霉病菌侵染的影响

 番茄灰霉病菌(Botrytes cinerea)的分生孢子萌发需要高湿的环境,一般要求相对湿度在80%以上或水滴中萌发最好。在适温下,随着湿度的增加,病菌致病性增强,孢子在相对湿度85%以上、菌丝在相对湿度80%以上的高湿环境能够发生侵染,并形成危害性的软腐斑。在一天中高湿的时间直接影响番茄灰霉病的发生,高湿(相对湿度超过85%)时间8h以上病菌才能够连续侵染。     

Influence of environmental conditions on dispersal of Botrytis cinerea conidia and on post-harvest infection of gerbera flowers grown under glass

Dispersal of Botrytis cinerea in a gerbera crop grown in two glasshouses 30 km apart was studied over a period of 18 months, in 1988 and 1989. Conidia were caught in spore traps consisting of agar in petri dishes exposed at different heights in the crop in each glasshouse. No seasonal patterns could be identified in the spore catches, assessed as colonies on the agar traps after incubation. The number of lesions caused by conidial infection of gerbera flowers following incubation, however, showed a distinct pattern. In spring and early summer few lesions were recorded whereas at other times of the year many lesions appeared. In linear regression analysis, variation in numbers of colonies (spore catches) could not be explained by environmental factors recorded during the experiments. Linear regression accounted for 77% and 81% of the variation in the number of lesions on flowers in the two glasshouses, in terms of relative humidity (postively correlated), global radiation outside the glasshouse (negatively correlated) and age of the crop (positively correlated). Despite differences in the systems by which the gerbera crop was produced and in the spore catches, the numbers of lesions on gerbera flowers in the two glasshouses were significantly correlated though not significantly different from each other.     

喜树碱对番茄灰霉病菌的病理反应

以番茄灰霉病菌为供试菌种,采用电导率法、吸光度法和电镜分析法对经过喜树碱（CPT）处理的番茄灰霉病菌进行病理反应研究。试验结果表明经200 mg/L CPT溶液处理后,番茄灰霉病菌菌丝细胞膜的通透性增加了30.7%,细胞外蛋白质浓度提高了13.5%。番茄灰霉病菌经CPT溶液处理后,还原糖和可溶性蛋白的含量随着处理时间的增长先升高后降低最后趋于平缓,在0～1 h之间增长最快。CPT处理后的番茄灰霉病菌的菌丝畸形,出现膨大或缢缩,菌丝生长点缢缩,细胞中的空腔增多,线粒体增多且线粒体嵴变得模糊,细胞核有降解的现象。     

武夷菌素对番茄灰霉菌(Botrytis cinerea)的作用方式

 借助光镜和透射电镜观察了武夷菌素对番茄灰霉菌菌丝生长和分生孢子萌发的形态影响,结果表明,武夷菌素处理的菌丝异常生长,分生孢子萌发的芽管膨大缢缩,菌丝体内液泡增多;武夷菌素能引起菌丝细胞膜透性的变化,造成液体培养基电导率增加;同位素标记的¹⁴C-谷氨酸、³H-葡糖胺和³H-腺嘌呤掺入实验表明,武夷菌素能抑制菌丝蛋白质的合成,对细胞壁几丁质和核酸的合成没有影响。     

Effects of certain herbicides on the oxidative decarboxylation of tryptophan by peroxidase

Non-enzymic and peroxidase-catalysed oxidative decarboxylations of tryptophan (TPP) were studied. The in-vitro rate of enzymic reaction was affected by various herbicides at low concentrations (10^?5?10^?4_M): dinoseb, 2,4-D, dichlobenil and benazolin acted as inhibitors; on the other hand, chlorpropham, bromacil, diphenamid and 4-ethylamino-6-isopropylamino-1,3,5-triazin-2-ol (hydroxyatrazine) were stimulatory. The results of in-vivo experiments showed that chlorpropham and 2,4-D changed the activity of peroxidase from Brassica germinated seeds in vivo, as they do in vttro. Determinations of consumed TPP were carried out either by spectro-photometry or by chromatography.     

灰霉菌(Botrytis cinerea)采后致病性研究

 灰霉菌(Botrytis cinerea)是引起葡萄采后病害的主要病原真菌之一。葡萄灰霉菌可在田间潜伏侵染,采后由健康果实携带进入销售市场,该菌的显著致病症状为果实软腐和脱落。灰霉菌与葡萄的其它采后致病菌,如链格孢菌(Alternaria alternata)、镰刀菌(Fusarium sp.)、芽枝霉(Cladosporium sp.)、青霉菌(Penicillium sp.)、黑曲霉(Aspergillus nigar)和粉红单端孢菌(Trichothecium roseum)相比,不仅表现出明显的潜伏侵染优势,而且具有较强的低温(4℃)条件下的致病优势。4℃低温下灰霉菌在寄主葡萄体外和体内分泌多聚半乳糖醛酸酶(PG)活力均显著高于以上各菌,而在25℃下无显著差异,这一结果与该2种温度下灰霉菌接种果实后的症状表现一致。     

New aspects on the infection mechanism of Botrytis cinerea Pers

With the help of active types of oxygen, extracellular enzymes and mechanical processes,Botrytis cinerea is capable of infecting plant tissue. Whereas no clear correlation was found between the activities of protease, pectolytic or other cell wall degrading enzymes and virulence, a positive correlation between pathogenicity and the intensity of active types of oxygen released was apparent in some isolates. It is assumed that these toxins result from the activity of glucose or xylose oxidases. Antioxidants inhibited the infection. It is concluded from these results, that active types of oxygen play a decisive role in the infection process.     

灰霉菌的抗药性与适合度测定

室内监测了北京、沈阳、保定等地区番茄灰霉菌 (Botrytis cinerea)对不同杀菌剂的抗药性 ,发现抗多菌灵菌株占监测总数的 74 .6 7% ,抗乙霉威菌株占 2 5.82 % ,还监测到 3株抗多菌灵、乙霉威菌株——即双抗菌株 ;利用菌落直径法和番茄叶片法测定了 3种抗药类型的番茄灰霉菌在离体和活体条件下的适合度 ,结果表明 ,不同抗药类型菌株在生长速率、致病力、繁殖力等方面差异不明显。     

Antifungal vapour-phase activity of allyl-isothiocyanate against Penicillium expansum on pears

The fungitoxicity of allyl-isothiocyanate (AITC) vapour against Penicillium expansum , the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L⁻¹ AITC-enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 10³ conidia mL⁻¹ to almost 80% at 1 × 10⁶ conidia mL⁻¹. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole-resistant strain of P. expansum , reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum .     

Ultrastructural study on the interaction between Physcomitrella patens and Botrytis cinerea

Little is known about the interactions of bryophytes with their pathogens compared with those of flowering plants. This study used the moss Physcomitrella patens and fungal pathogen Botrytis cinerea to investigate their interaction at the ultrastructural level. Infection behaviour of B. cinerea in moss tissues was observed for 1–7 days after inoculation. Some appressoria directly penetrated P. patens cytoplasm through leaf cell walls, and others entered the intercellular spaces of leaves and infected cytoplasm. Infection hyphae were observed in sclerenchyma cells of P. patens stems for the first time. The results demonstrated that the ratio of hyphae‐inoculated leaf cells to leaf numbers could be used to quantify the disease development process at 24 h after inoculation. The ultrastructural studies revealed two responses of P. patens to B. cinerea inoculation: reinforcement of cell walls, including papillae formation; and cell death. This was the first observation of papillae for P. patens–B. cinerea interaction at the ultrastructural level. Papillae in P. patens had a similar ultrastructure to those of higher plants, and may be involved in the defence response to B. cinerea infection. Thus papillae formation is probably an evolutionarily conserved defence mechanism from the early land colonization by plants. Cell death during the P. patens–B. cinerea interaction had some features of programmed cell death, with hydrogen peroxide produced in cytoplasm membranes, suggesting something like a hypersensitive response. These ultrastructural studies suggest that P. patens could be a useful system for studying phytopathogens and could provide theoretical bases for defence mechanisms in evolutionary development.