EFFECT OF CERTAIN CHLORONITROBENZENES ON GERMINATION, GROWTH AND SPORULATION OF SOME FUNGI

When grown in an atmosphere saturated with tetrachloronitrobenzene (TCNB) vapour, Botrytis cinerea showed retarded germination and colony growth and its sporulation was completely suppressed. Retardation of growth was greater on dilute or acidified media and at lower temperatures.
Similar effects were shown by Bhizoctonia solani, Phytophthora parasitica, Mucor hiemalis and Trichoderma viride but none of these was as sensitive as Botrytis cinerea , especially as regards linear growth. Of two strains of Fusarium caeruleum , one was much more sensitive than the other, both in linear growth and sporulation. Pythium deBaryanum was unaffected in its growth rate.
TCNB, as a growth repressant, is fungistatic without being fungicidal.
With fungi which are sensitive, tetrachloronitrobenzene was more effective than the penta-derivative, except for Trichoderma viride where the converse applied.     

Characterisation of acetaldehyde effects on Rhizopus stolonifer and Botrytis cinerea

Acetaldehyde (AA) vapour reduced the in vitro growth of the fruit-infecting fungi Botrytis cinerea and Rhizopus stolonifer, and decreased the respiration of R. stolonifer. Spore germination, germ tube elongation and sporulation were inhibited in both fungi. AA induced the leakage of electrolytes from B. cinerea and R. stolonifer membranes. The conductivity found in the fungal extracts was positively correlated with the AA concentration applied. The leakage of reducing sugars and amino acids suggest that cell membranes are irreversibly disrupted by AA as a first step in inhibition of the fungi in vitro.     

Sporulation of Bacillus subtilis in Continuous Culture

Sporulation of Bacillus subtilis 168 was studied in chemostat cultures. Sporulation occurred at high frequency under limitation of growth by glucose or the nitrogen source in minimal medium, whereas rates of sporulation were low for Mg(2+), phosphate, citrate, or tryptophan limitation. Sporulation was found at all growth rates tested, and the incidence of spores increased with decrease in growth rate of the culture. Within the range of growth rates up to the maximum obtainable with the defined medium, no threshold effect of growth rate on sporulation was observed. By studying transient states, it was possible to determine the time taken for the appearance of a refractile spore after initiation of a cell to sporulation. Under conditions of glucose limitation, cells were found to be committed to sporulation as soon as they were initiated. In nitrogen-limited cultures, however, a partial relief of nitrogen limitation prevented the development of spores during the first hour after initiation. The results of experiments with multistep changes in dilution rate of a chemostat culture indicate that initiation to sporulation is probably restricted to a particular point in the cell division cycle.     

Ability of Clonostachys rosea to Establish and Suppress Sporulation Potential of Botrytis cinerea in Deleafed Stems of Hydroponic Greenhouse Tomatoes

The ability of Clonostachys rosea to establish and persist in deleafed tomato stems and to suppress sporulation potential of Botrytis cinerea was investigated in plots of hydroponic tomatoes in commercial greenhouses. Leaves near lower fruit clusters were removed according to standard practice and deleafed portions of the stems were treated with C. rosea , iprodione or water. Inoculum of B. cinerea was from natural infections. Stem lesions were not produced by the pathogen during the trials. Development of C. rosea and B. cinerea in stems was estimated indirectly by quantifying sporulation on excised stem tissues that were incubated on an agar medium containing paraquat. Incidence and area of sporulation of C. rosea on tissue pieces were high (76-99%) and moderately high (33-79%), respectively, when stems were treated with the agent at 0, 6, 24 or 48 h after deleafing and sampled 11 to 75 days later. In various instances, the agent also sporulated on tissues from water controls and iprodione treatments, apparently after interplot transmission. In most instances, incidence and area of sporulation of B. cinerea on tissue pieces were high (83-100%) and moderate to high (35-76%), respectively, in the water controls, but moderate (31-44%) and moderate to low (5-34%), respectively, for stems treated with C. rosea at 0 to 48 h after deleafing and sampled after 11-75 days. Without exception, C. rosea suppressed B. cinerea as or more effectively than iprodione. Correlations between inoculum density of C. rosea (0-10 6 conidia mL -1 ) and sporulation potential of B. cinerea in deleafed stems were strongly negative in each of three tests ( r = -0.95 to -0.99). Conidial suspensions and a talc formulation of C. rosea were of similar effectiveness against B. cinerea . We conclude that C. rosea persisted and suppressed sporulation potential of B. cinerea in deleafed tomato stems for at least 11 weeks after application.     

Suppression of Botrytis cinerea sporulation by Clonostachys rosea on rose debris: a valuable component in Botrytis blight management in commercial greenhouses

Botrytis blight, caused by Botrytis cinerea (Bc), is an important disease on roses grown in plastic greenhouses in Brazil. Biocontrol with Clonostachys rosea (Cr) applied to leaves and crop debris to reduce pathogen sporulation can complement other control measures for disease management. Two experiments, each with a rose cultivar, were conducted in a plastic greenhouse. For ‘Red Success,’ four treatments were compared: (1) control; (2) fortnightly sprays of Cr; (3) weekly sprays of mancozeb; and (4) weekly sprays of either Cr or mancozeb to the lower third of the plants and the debris. For ‘Sonia,’ treatment 4 was not included. Samples were taken from debris (leaves and petals) at ten 15-day intervals and plated on PCA medium. Sporulation of fungi and incidence of Botrytis blight on buds were assessed. For both cultivars, C treatments significantly (P=0.05) reduced Bc sporulation. However, disease incidence was not consistently reduced, probably because the applications of C. rosea started when Botrytis blight epidemic was advanced and no sanitation practices were performed on nontreated plots. From the present and previous studies, continuous application of Cr on debris, associated with sanitation practices, has the potential to reduce Bc sporulation and disease incidence in the buds.     

灰葡萄孢犬尿氨酸途径关键酶基因的鉴定

【背景】灰葡萄孢是一种重要的植物病原真菌,实验室前期明确了灰葡萄孢犬尿氨酸单加氧酶(kynurenine3-monooxygenase,KMO)基因BcKMO参与调控病菌的生长发育和致病力。犬尿氨酸单加氧酶(KMO)是犬尿氨酸途径的关键酶,但灰葡萄孢是否存在犬尿氨酸途径及其在病菌生长、发育和致病过程中的功能尚未见相关报道。【目的】鉴定灰葡萄孢犬尿氨酸途径中的关键酶基因,确定灰葡萄孢犬尿氨酸途径的存在,为阐明灰葡萄孢生长发育和致病力的分子机理奠定基础。【方法】利用生物信息学方法,对灰葡萄孢犬尿氨酸途径中犬尿氨酸酶(kynureninase,KYN)、吲哚-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)、犬尿氨酸氨基转移酶(kynurenine amino transferase,KAT)的编码基因进行分析;利用Real-time PCR技术,检测灰葡萄孢野生型BC22、BcKMO基因T-DNA插入突变体BCG183、恢复菌株BCG183/BcKMO中犬尿氨酸途径关键酶基因的表达水平;利用真菌犬尿氨酸酶KYN检测试剂盒,测定BcKMO突变体中犬尿氨酸酶(KYN)的含量。【结果】灰葡萄孢中含有2个犬尿氨酸氨基转移酶(KAT)的编码基因、3个吲哚-2,3-双加氧酶(IDO)的编码基因、10个犬尿氨酸氨基转移酶(KAT)的编码基因。灰葡萄孢KYN编码基因、IDO编码基因、KAT编码基因在突变体BCG183中的表达水平显著高于或低于在野生型和恢复菌株。突变体BCG183中犬尿氨酸酶(KYN)的含量显著低于野生型BC22和恢复菌株。【结论】灰葡萄孢中存在犬尿氨酸途径,灰葡萄孢BcKMO基因突变影响KYN、IDO和KAT编码基因的表达以及犬尿氨酸酶(KYN)的含量。     

酶法制备壳寡糖对番茄灰霉病病菌抑制机理初探

目的：研究酶降解法制备的壳寡糖对番茄灰霉病病菌的抑制机理。方法：测定壳寡糖对番茄灰霉病菌菌丝发育、孢子萌发的抑制作用以及对菌丝形态、细胞膜透性和对菌丝体可溶性蛋白质含量的影响。结果：当壳寡糖处理浓度为1.5mg/mL以上时,对菌丝生长的抑制率达70%以上,EC50为0.72mg/mL。当处理浓度为6mg/mL时,对分生孢子的抑制率显著优于其他浓度水平,达87.4%,EC50为1.48mg/mL。显微观察,浓度为0.8mg/mL的壳寡糖处理可诱导菌丝分枝增多、菌体分隔增加,分生孢子形成受到抑制。此外壳寡糖能够引起菌丝细胞膜透性发生变化,造成菌丝体内含物的渗漏。壳寡糖处理66 h后,菌丝体可溶性蛋白含量比对照降低了36.7%。结论：壳寡糖对番茄灰霉病病菌抑制机理的探究为研究壳寡糖这一新型生物农药的作用机制提供依据。     

Effect of light quality on the photoinduction of carotenoid synthesis in Verticillim agaricinum

The action spectrum of photoinduction of carotenoid biosynthesis in Verticillium agaricinum has a high peak in the near-UV region with very small peaks in the blue, yellow and red regions. This suggests that neither a flavoprotein nor a carotenoid could be the photoreceptor, but a new unknown pigment system. Sporulation however is not photoregulated and is unaffected by light in the near-UV region. So, mycochrome cannot be implicated in the sporulation of this fungus. Further, sporulation and carotenogenesis must be under separate regulatory control in this organism, but not necessarily so in other organisms.     

The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea

AIMS: To establish whether germination of Botrytis cinerea was affected by the symbiosis of Bacillus subtilis L-form bacteria with Chinese cabbage. METHODS AND RESULTS: Germinating seeds of Chinese cabbage were co-cultivated with either L-forms of Bacillus subtilis or 5% (w/v) mannitol by soaking for 3 h. Seeds were then washed in sterile water, sown on a minimal medium and incubated in controlled conditions. L-form symbiosis was detected over a time course by ELISA. Conidial germination of Botrytis cinerea was significantly reduced on cotyledonous leaves of L-form-treated plants compared with controls. CONCLUSIONS: Symbiosis of B. subtilis L-form bacteria during seed germination of Chinese cabbage inhibits conidial germination in plants on subsequent exposure to Botrytis cinerea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first account of plant symbiosis with L-form bacteria showing antagonism to a fungal plant pathogen. This has promising implications for the use of this L-form as a biocontrol agent.     

Characterization of a new sporulation factor in Bacillus subtilis.

We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density. Proline or arginine is required for stimulation under the conditions of our assay. Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase. It is required in small amounts and is resistant to various proteolytic agents. Several spo mutants were tested for the ability to produce functional sporulation factor. All of these mutants produce factor and do not sporulate in the presence of factor from wild-type cells. Sporulation factor is not involved in the induction of alpha-amylase synthesis at the initiation of sporulation.     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: I. EFFECT OF MAGNESIUM AND CALCIUM IONS

Sporulation in a strain of the wild yeast, Hansenula saturnus,was investigated. The yeast was found to form spores even indistilled water. The sporulation rate (percentage of ascus-bearingindividuals) in this case was found to be markedly affectedby the cell concentration adopted in the test. The addition of inorganic nutrients to the sporulation mediumstimulates sporulation. The yeast requires either magnesiumor calcium for growth and sporulation. Higher concentrationsof these ions are required for sporulation than for growth.In both cases magnesium is effective at more dilute concentrationsthan calcium. Under the conditions of the experiments, in which the yeastforms a pellicle, the sporulation rate in the pellicle far exceedsthat in the sediment. The effects of environmental factors on the sporulation wasconsidered in relation to growth. It was found that, under theconditions of poor growth in the sporulation culture, no exogenousmagnesium and calcium are required for sporulation. In suchcases, the yeast cells are inferred to have an endogenous stockof magnesium and calcium enough for the sporulation. ¹ Present address: Laboratory of Microbiology, Department ofAgriculture, Tôhoku University, Sendai. (Received May 4, 1961; )     

Biocontrol agents of Botrytis cinerea tested in climate chambers by making artificial infection on tomato leafs

To reduce the use of chemical agents, that are causing damage to the environment, in the fight against Botrytis cinerea, different BCA's were tested for their possibility to control Botrytis cinerea in a biological way. In order to investigate the effectiveness of the different micro organisms and Elot-Vis, experiments were carried out in climate chambers with 5 weeks old tomato plants. Leafs on the plant were inoculated with drops of a suspension that contained spores of Botrytis cinerea. The possible antagonists that were tested in these experiments were Trichoderma harzianum (Trichodex), T. asperellum (Biofungus), T. hamatum (T382), Bacillus subtilis (Serenade and Phytovit) and Pseudomonas aeruginosa (7NSK2 and KMPCH). For all these different micro organisms the direct and the indirect influence on Botrytis cinerea was investigated. In tests where the direct influence of the antagonists was examined, the spores of the moulds or the bacteria were suspended together with spores of Botrytis cinerea and subsequently drops of this suspension were pippeted on the leafs. After a while by ideal circumstances for Botrytis cinerea the infections on the inoculated leafs were counted. For the indirect influence of the antagonists, also leafs of 5 weeks old tomato plants were inoculated with a suspension of Botrytis cinerea spores. The roots of the tomato plants that were used for testing the indirect influence were treated during there growth with a suspension of the antagonist to see if induced systemic resistance pathway (ISR) was activated. For testing the effectiveness of Elot-Vis, tomato plants were sprayed a few times with a solution of this product during their growth. Results of the climate chamber test of the plants that were treated with Elot-Vis, showed a reduction of Botrytis cinerea infections on the inoculated leafs. Biological control agents seem to be not always very effective against Botrytis cinerea. The biological control agents that are containing micro organisms are also depending on the circumstances of the environment for an optimal development. These conditions are not always that optimal to compete with Botrytis cinerea or other micro organisms that are present on the plant.     

Different products for biological control of Botrytis cinerea examined on wounded stem tissue of tomato plants

For the moment the agents that are used against Botrytis cinerea, in glasshouses were tomatoes are cultivated, are from chemical origin. For reducing the use of chemical agents in the future it is important to search for effective biological control agents against the fight of Botrytis cinerae. The following biological products Vital pasta, Vital gel and Elot-Vis were examined in there possibility to control Botrytis cinerea. Elot Vis was tested out in experiments that were carried out in climate chambers were leafs of 3 week old tomato plants were artificially infected with Botrytis cinerea spores. Also the biological products of Vital were first investigated in experiments that were carried out in climate chambers. In stead off leafs of tomato plants it were stem wounds of tomato plants who were treated with the pasta or the gel that was spread over the wounded surface after this has been inoculated with a suspension of conidia of Botrytis cinerae. The results of these first tests that were executed in the climate chambers were the circumstances for Botrytis cinerea were ideal seemed promising. In the next step these products were tested out on large scale in glasshouses. For each plant 5 wounds were created by removing the leafs, these wounds were or first treated with the Biological product and thereafter artificially infected with Botrytis cinerea spores to check out if these products can be used as a preventive agent or the wounds were first inoculated with a suspension of Botrytis cinerea spores and thereafter treated with the product. For the product Elot-Vis a few plants were totally sprayed with an Elot-Vis suspension before leafs were removed and the wounds were inoculated with conidia of Botrytis cinerea to check out if this product was able to activated the induced systemic resistance pathway. The experiments that were executed in glasshouses showed different percentages of succeeded Botrytis cinerea infections. This is probable due to the different weather conditions during both days that the experiments were executed. For the wounds that were treated with pasta it was difficult to distinguish wounds were Botrytis cinerea succeeded to infect the plant, because these wounds frequently didn't show any sign of infection on the surface but when the wounds with the pasta were cut open it was possible to see Botrytis cinerea infections inside the stem.     

Transformations of testosterone and related steroids by Botrytis cinerea

Botrytis cinerea (strain AM235) was used to investigate the transformations of testosterone and related steroids. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. Botrytis cinerea converts the examined substrates mainly to 7 alpha-hydroxy derivatives. 1-Dehydrotestosterone was also significantly hydroxylated at a 14 alpha-position.     

生防细菌CNY-04筛选、鉴定及其对灰霉病菌的防效

【目的】为研究土壤细菌对蔬菜灰霉病的生防价值, 从辽宁、山东等地区的蔬菜种植基地采集土壤样本56份, 分离、筛选出对灰霉病具有稳定拮抗作用的细菌9株。【方法】采用平板对峙培养法进行初筛、复筛, 用抑菌圈法测定其抑菌效果, 并进行离体果实试验验证其对蔬菜灰霉病的防治效果, 通过形态学特征、生理生化特征及16S rRNA基因序列分析研究其分类地位。【结果】细菌CNY-04对蔬菜灰霉病的拮抗能力最强且遗传稳定, 抑菌圈直径达到34 mm; 初步鉴定该菌株为格氏沙雷菌(Serratia grimesii), 尚未见该菌在生防上的报道; CNY-04液体菌剂对离体番茄果实灰霉病的防效为69.23%, 50%多菌灵防效为75.39%, 24 h时接种CNY-04处理的番茄发病率为40.0%, 而48 h时接种处理的发病率为51.1%。【结论】CNY-04是一株较为理想的拮抗菌, 丰富了生防资源。     

HM17, a new polyene antifungal antibiotic produced by a new strain of Spirillospora

H. HACÈNE, K. KEBIR, D. SID OTHMANE AND G. LEFEBVRE. 1994. An antifungal antibiotic (HM17) was obtained from a new isolate classified to the genus Spirillospora on the basis of its chemical and morphological properties. On solid media this antibiotic strongly inhibited the growth of strians of Fusarium oxysporum formae speciales albedinis, Botrytis cinerea, Gaeumaniomyces graminis and several other fungi known to be plant and human pathogens. Antifungal activity in culture collection strains of Spirillospora has not so far been reported. The u.v. absorption spectrum and physico-chemical characteristics place HM17 in the methylpentaene sub-group of polyene macrolides. HM17 is different from other known methylpentaenes. This is the first report of polyene production by a Spirillospora.     

Intracellular reactive oxygen species mediate suppression of sporulation in Bacillus subtilis under shear stress

Sporulation is an important cellular response to stress that is also significant from a bioreactor operation viewpoint. While sporulating organisms are known to show an enhanced sporulation response under several stress situations, the sporulation response to shear stress has not been investigated thus far. Such a study could be of interest since shear stress, to a greater or lesser degree, is always present in bioreactor operation. In this article, we investigate the sporulation extents of the Gram-positive bacteria Bacillus subtilis at various defined shear levels. We show that, contrary to expectations, shear inhibits sporulation. We found an inverse correlation between the shear rate-dependent specific intracellular reactive oxygen species level (siROS), and the sporulation extent. A 10-fold increase in siROS resulted in about 17-fold decrease in sporulation extent. The involvement of reactive oxygen species (ROS) in sporulation was unknown thus far. Further, through experiments that specifically increased and reduced intracellular ROS (iROS), we established that siROS is responsible for the inhibition of sporulation under shear stress. In addition, we found that shear induced siROS regulated the expression levels of the general stress proteins Ctc and sigma(B). Based on the above, we hypothesize that siROS may regulate suppression of sporulation under high shear by altering sigma(B) and Ctc expression levels, and a model for the same is presented.     

FACTORS CONTROLLING THE SPORULATION OF YEASTS. I. THE PRESPORULATION PHASE

SUMMARY: Yeasts tend to dissociate into mixtures of cell types with different powers of sporulation; hence single cell isolates are recommended for sporulation studies. The ability of yeasts to produce 4-spored asci can be improved by single cell selection. Cells from actively fermenting cultures sporulate much better than those grown under aerobic conditions. Sporulating ability depends on fermentation 'age', reaching a maximum when 85–90% of the CO₂ has been evolved. Carbon dioxide assimilation in the presporulation phase appears essential for maximal sporulation, but complete anaerobiosis in this phase is detrimental to sporulating ability. Malt wort cultures of a baker's yeast have given remarkably constant figures, in successive tests, for sporulation; but some batches of wort have an adverse effect on sporulating ability. The same yeast, grown on Lodder-Rij's synthetic medium containing 4 or 8% (w/v) of glucose, is capable of 80% sporulation (proportion of cells forming asci) on sodium acetate agar, comparable to that obtainable with malt wort cultures. Sporulation is depressed by excess storage of fat, while storage of glycogen does not affect sporulating ability.     

Seasonal Variation in Sporulation of Phytophthora infestans

Sporulation ability of two isolates of Phytophthora infestans maintained on potato tuber slices of a susceptible cv. ‘Bintje’ and on-rye agar medium was studied for four years (1981–1984). This feature of the fungus was shown to vary in particular seasons during the year. Significantly higher sporulation density per cm² of aerial mycelium on potato tuber slices was observed in winter and late autumn while significantly lower sporulation was found in spring. Similar tendencies were observed when one of the isolates was cultivated on rye agar medium under controlled conditions. Positive correlation was found between sporulation patterns of isolates of the fungus maintained on rye agar and on tuber slices. Hypothesis has been proposed that these changes are due, to a biorhythm in the fungus.     

Control of sporulation in the filamentous cyanobacteriumAnabaena torulosa

In the cyanobacteriumAnabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculatedA. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.