Ultrastructural changes observed in Colletotrichum alienum conidia following treatment with cold plasma or plasma-activated water

Previous studies demonstrated that a reduction in germination of Colletotrichum alienum conidia could be achieved following treatment with cold plasma (CP) or incubation with plasma-activated water (PAW). In this study, the mode of action of CP and PAW on C. alienum conidia was explored using transmission electron microscopy. Following treatment of the conidia, noticeable ultrastructural changes were observed, including cell wall maceration, disorganization of the cytoplasm and vacuole, and changes to the nuclei and mitochondria. Disorganization of the cytoplasm was exhibited by 85% of conidia after CP or PAW treatment. Also, 85% of conidia were observed with disorganized vacuoles after 6 min of CP treatment and 65% after 3 hr incubation in PAW. Further, 45% or more conidia had modified cell walls after PAW incubation or CP treatment. Deformation of conidia was observed in 23% of conidia after PAW incubation. These ultrastructural changes are a likely reason for the reduced germination of C. alienum conidia following CP or PAW treatment.     

Cold plasma as a novel treatment to reduce the in vitro growth and germination of Colletotrichum species

This study investigated the use of cold plasma to reduce the in vitro growth of two postharvest fungal plant pathogens, Colletotrichum alienum and C. fioriniae, isolated from avocados. Cold plasma (CP) was used to treat pure cultures and conidial suspensions of both pathogens, for 180 or 360 s, in either open or sealed environments from varying distances. In an open environment, the 360 s treatment at a distance of 5 cm reduced the colony growth of freshly inoculated cultures to less than 2 mm/day compared to the control of more than 8 mm/day, and treatment of conidial suspensions resulted in almost 100% reduction of conidial germination. In the same environment, the 180 s CP treatment did not significantly reduce the colony growth of fresh or actively growing cultures, but did suppress the germination of conidia by up to 80%. In a sealed environment, the 360 s CP treatment also effectively reduced the growth of freshly inoculated cultures, with no growth for some isolates. Production of reactive oxygen and nitrogen species was observed during treatment, and these may have contributed to the reduction in growth and germination. These results demonstrate the potential of CP for the control of two Colletotrichum species.     

The effect of temperature and water potential on the production of conidia by sclerotia of Botrytis squamosa

The rate of conidiogenic germination of Botrytis squamosa was highest at 16°C and the greatest numbers of conidia per sclerotium (up to 5 × 10⁴) were produced at temperatures of 5–10°C. At temperatures above 20°C, the percentage of sclerotia producing conidia declined rapidly. Decreasing water potential reduced the rate at which conidia were produced and also resulted in fewer conidia produced per sclerotium. However, conidia were produced at water potentials as low as −2 MPa, at which sclerotial germination was at least 60%. A simulation model that included effects of both temperature and water potential was developed from laboratory and field data obtained for conidial production in sclerotia exposed for periods of 1, 2, 3 or 4 weeks during an entire year. There was good agreement between conidiogenic germination predicted by the model and conidial production observed in onion plots artificially inoculated with sclerotia. Temperature and water potential were therefore considered to be the principal microclimatic factors affecting conidial production by B. squamosa. The role of sclerotia in the context of UK onion production is discussed.     

Effect of hot water treatment on peach volatile emission and Monilinia fructicola development

The effect of hot water treatment (HWT) to control peach brown rot was investigated. Peaches were dipped in water at 60°C for 60 s and artificially inoculated with Monilinia fructicola conidia. HWT failed to control brown rot if applied before inoculation and microscopic observations revealed a stimulatory effect on germ tube elongation of M. fructicola conidia placed immediately after HWT on the fruit surface, compared to the control. The influence of fruit volatile emission due to HWT was performed on the pathogen conidia exposed to the headspace surrounding peaches. The results showed an increase of M. fructicola conidial germination ranging from 33 to 64% for cultivars Lucie Tardibelle and Red Haven heat‐treated peaches, respectively, compared to the control. The volatile blend emitted from heat‐treated fruit was analysed by solid‐phase microextraction/gas chromatography‐mass spectrometry (SPME/GC‐MS) and proton transfer reaction‐time of flight‐mass spectrometry (PTR‐ToF‐MS). Fifty compounds were detected by SPME/GC‐MS in volatile blends of cv. Lucie Tardibelle peaches and significant differences in volatile emission were observed among heated and control fruit. Using PTR‐ToF‐MS analysis, acetaldehyde and ethanol were detected at levels 15‐ and 28‐fold higher in heated fruit compared to unheated ones, respectively. In vitro assays confirmed the stimulatory effect (60 and 15%) of acetaldehyde (0·6 μL L^?1) and ethanol (0·2 μL L^?1) on M. fructicola conidial germination and mycelial growth, respectively. The results showed that volatile organic compounds (VOCs) emitted from heat‐treated peaches could stimulate M. fructicola conidial germination, increasing brown rot incidence in treated peaches when the inoculation occurs immediately after HWT.     

马铃薯早疫病菌分生孢子萌发条件的研究

为了明确马铃薯早疫病菌分生孢子萌发的条件,研究了温度、湿度、pH、光照、营养条件等对马铃薯早疫病菌分生孢子萌发的影响。结果表明,分生孢子在水滴中于30℃1 h即可萌发,8 h后达到萌发高峰,萌发率为98.4%;萌发的最适温度为30℃;相对湿度85%以上均可萌发,水滴中萌发率最高;pH6~10的条件利于分生孢子萌发;紫外线对分生孢子萌发有抑制作用;无机氮源中的铵态氮和尿素对孢子萌发有抑制作用,有机氮源中半胱氨酸和谷氨酸对孢子萌发有抑制作用;山梨糖不利于分生孢子萌发;光照对分生孢子萌发也有抑制作用;分生孢子致死温度是57℃。     

Effects of temperature and leaf age on conidial germination and disease development of powdery mildew on rubber tree

Powdery mildew is an important disease of rubber trees worldwide. To assess the effects of temperature and leaf age on conidial germination and disease development, conidia were inoculated onto rubber tree seedlings with leaves at three phenological stages (copper bronze, colour-changing, and light green) and then incubated at six constant temperatures (10, 15, 20, 25, 30, and 35°C). Leaf age did not affect conidial germination (p = .296) whilst temperature did (p < .0001), although conidia were able to germinate at all tested temperatures. The estimated optimal temperature for conidial germination was 23.2°C. Leaf age, temperature, and their interactions had significant effects on conidial infection and hypha number (p < .0001). At 10 and 35°C, more than 2 and 4 days were needed for infection to complete, respectively, compared to <2, 1, 0.5, and 0.5 days for 15, 20, 25, and 35°C, respectively. Sporulation and mildew symptoms were only observed on those inoculated leaves of all stages at 20 and 25°C, and at the copper bronze stage only at 15°C. The latent period on the copper bronze leaves at 15°C was longer (9 days) than at 20 and 25°C (4 days). The latent period at 20 and 25°C increased from 4 to 7 days as the leaf development stage increased from copper bronze to light green. Therefore, temperature affected germination and postgermination growth of rubber tree powdery mildew, whereas leaf age primarily affected postgermination growth of the pathogen.     

Production, Survival, and Evaluation of Solid-Substrate Inocula of Penicillium oxalicum, a Biocontrol Agent Against Fusarium Wilt of Tomato

ABSTRACT Production of conidia of Penicillium oxalicum (ATCC number pending), a biocontrol agent of Fusarium oxysporum f. sp. lycopersici, was tested in liquid and solid fermentation. P. oxalicum produced 250-fold more conidia in solid than in liquid fermentation at 30 days after inoculation of substrate. Solid fermentation was carried out in plastic bags (600 cm(3)) especially designed for solid fermentation (VALMIC) containing 50 g of peat/vermiculite (PV) (1:1, wt/wt) with 40% moisture, sealed, sterilized, and then inoculated with 1 ml of a conidial suspension of P. oxalicum (10(5) conidia g(-1) dry substrate), sealed again, and incubated in darkness at 20 to 25 degrees C for 30 days. Addition of amendments to PV in a proportion of 0.5 (wt/wt) significantly increased conidial production of P. oxalicum. The best production was obtained on PV plus meal of cereal grains (barley) or leguminous seeds (lentil) (100-fold higher). Conidial production obtained after 5 days of inoculation was similar to that obtained at 30 days. However, viability of conidia produced in PV plus lentil meal was 35% higher than that of conidia produced in PV plus barley meal. Changes in proportions (1:1:0.5, wt/wt/wt; 1:1:1, wt/wt/wt; 1:0.5:0.5, wt/wt/wt; 1:1:0.5, vol/vol/vol) of components of the substrate (peat/vermiculite/lentil meal) did not enhance production or viability of conidia. Optimal initial moisture in the substrate was 30 to 40%. At lower moistures, significant reductions of production of conidia were observed, particularly at 10%. There was a general decline in the number of conidia in bags with time of storage at -80, -20, 4, and 25 degrees C, or at room temperature (range from 30 to 15 degrees C), with the highest decline occurring from 60 to 180 days. Conidial viability also was reduced with time, except for conidia stored at -20 degrees C. Fresh conidia produced in solid fermentation system or those conidia stored at -20 degrees C for 180 days reduced Fusarium wilt of tomato by 49 and 61%, respectively.     

Influence of environmental factors on conidial germination and survival of Sphaeropsis pyriputrescens

Sphaeropsis pyriputrescens is the cause of Sphaeropsis rot in apples and pears. In this study, effects of temperature, wetness duration, relative humidity (RH), dryness, and interrupted wetness duration on conidial germination of the fungus were evaluated. Conidial germination and germ tube elongation occurred at temperatures from 0°C to 30°C. The optimum temperature for germination and germ tube elongation appeared to be 20°C, at which a minimum wetness period of 5 h was required. Conidia germinated at RH as low as 92% after 36 h at 20°C, but not at 88.5% RH. The effect of dry periods on germination depended on RH. Conidial germination at 85% RH was higher than that at 25% RH within a 4-h dry period, after which time no difference was observed. Less than 10% conidia germinated after a 10-day dry period at both 20°C and 28°C. Conidial germination decreased as the wetness duration prior to dryness increased. Conidia wetted for 6 h prior to dryness died within a 1-h dry period. After a 12-h dry period, no or few conidia germinated at 25% RH, whereas 3% to 10% of the conidia germinated at 85% RH and no further decrease was observed as the dry period increased. The results contribute to our understanding of conditions required for conidial germination of S. pyriputrescens and infection of fruit leading to Sphaeropsis rot.     

烟草米根霉对6种杀菌剂的敏感性及菌株保存条件筛选

采用孢子萌发法测定了烟草上米根霉Rhizopus oryzae对6种杀菌剂 (代森锰锌、多菌灵、嘧霉胺、嘧菌酯、啶酰菌胺及氟啶胺) 的敏感性，并就其适宜的保存条件进行了筛选，同时采用离体叶片法测定了上述6种杀菌剂对烟叶霉烂病的防治效果。结果表明:6种杀菌剂对米根霉孢子萌发和烟叶霉烂病均表现出了不同的抑制活性。其中，抑制孢子萌发活性最为明显的是氟啶胺和啶酰菌胺，其EC₉₀值分别为0.67 和1.53 mg/L；其次为代森锰锌和嘧菌酯，15.16和17.66 mg/L；最弱为嘧霉胺和多菌灵，71.87和81.96 mg/L。对烟叶霉烂病防效最好的为嘧菌酯，50 mg/L处理的防效为85%；其次为啶酰菌胺，200 mg/L处理时防效为83%；氟啶胺的防效较差，1 000 mg/L处理时仅为48%；而代森锰锌、多菌灵和嘧霉胺在最高使用剂量 (分别为4 000、800和800 mg/L) 时防效均低于20%。病原菌保存方法筛选结果表明，保存后米根霉的孢子萌发率均发生了不同程度降低。其中，4 ℃保存于20%甘油组的孢子悬浮液萌发率为60%；4 ℃保存的孢子干样萌发率为36%；4 ℃保存的孢子悬浮液和 –20 ℃保存于20%甘油的孢子悬浮液萌发率均低于20%；20 ℃保存的孢子干样萌发率为11%；–20 ℃保存的孢子悬浮液萌发率为6%。研究结果可为烘烤期烟叶霉烂病防治和米根霉孢子的保存提供参考依据。     

布朗李叶枯病菌生物学特性研究

对布朗李叶枯病菌(Macrophoma kawatsukai Hara)生物学特性研究表明,该菌最适生长和产孢的培养基为布朗李果汁。菌丝生长和产孢的最佳碳源分别为蔗糖、麦芽糖和果糖;氮源分别为酪氨酸、谷氨酰胺、天门冬酰胺和半胱氨酸。菌丝生长最适温度25℃,最适pH5～7;分生孢子形成最适温度25～30℃,最适pH8,饱和湿度有利于产孢,阳光照射形成分生孢子器最多,紫外光照射对分生孢子器形成有一定的抑制作用。孢子萌发最适温度为20～30℃,最适pH6～9,相对湿度为90%～100%,以在水滴中萌发最好。布朗李杆煎汁和布朗李果汁对孢子萌发有促进作用。     

Strawberry Plant Extracts Stimulate Secondary Conidiation by Colletotrichum acutatum on Symptomless Leaves

ABSTRACT Conidial suspensions of Colletotrichum acutatum were prepared in 1:27, 1:45, and 1:81 (wt/vol) dilutions of an extract of strawberry (cv. Tristar) flowers or leaves in water. Strawberry leaves and plastic coverslips were sprayed with the conidial suspensions, incubated at 25 degrees C and continuous wetness for 48 h, and the number of conidia and appressoria were counted. In another experiment, leaves and coverslips were sprayed with a conidial suspension in water, incubated for 72 h to establish C. acutatum populations, and placed in a growth chamber under dry conditions for up to 6 weeks. At each sampling time, leaves and coverslips were sprayed with flower extracts, leaf extracts, or water, incubated for 48 h at 25 degrees C and continuous wetness, and the number of conidia and appressoria were counted. Flower extracts significantly (P 相似文献   

Antifungal activities of hyoscyamine and scopolamine against two major rice pathogens: <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> and <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The antifungal activities of hyoscyamine and scopolamine, major alkaloids extracted from the desert plant Hyoscyamus muticus, against two rice pathogens, Magnaporthe oryzae and Rhizoctonia solani, were studied. The minimum inhibitory concentration of hyoscyamine that resulted in distinctive inhibition (MIC₅₀) was 1 μg/ml for both fungi. Exposure to hyoscyamine caused the leakage of electrolytes from the mycelia of both fungi. Hyoscyamine (>1 μg/ml) irreversibly delayed or inhibited conidial germination and appressorium formation in M. oryzae grown on polystyrene plates. Hyoscyamine effectively inhibited the attachment of conidia to the surface of rice (Oryza sativa) leaves and inhibited appressorium formation on the leaves. A high concentration of scopolamine (1000 μg/ml) also delayed or inhibited conidial germination in M. oryzae, but conidial germination was restored after washing the conidia with water. Antifungal activity of hyoscyamine was reduced by scopolamine. Magnaporthe oryzae infection was significantly suppressed (by >95%) in leaves of intact rice plants treated with hyoscyamine (10 μg/ml). Moreover, 10 μg hyoscyamine/ml significantly reduced the disease severity index for sheath blight to ≤0.2, when compared with the disease index of control plants (>7.0). Hyoscyamine (>20 μg/ml) completely inhibited sclerotial germination and development of R. solani by delaying the initiation, maturation, and melanization of the sclerotia. These results suggest that tropane alkaloids may be useful for controlling blast and sheath blight diseases of rice and for studying the mechanisms that regulate conidial germination in M. oryzae and sclerotial germination and development in R. solani.     

玉米大斑病菌孢子萌发和附着胞形成的影响因素研究

 Factors of influence on conidium germination and appressorium formation of Setosphaeria turcica,such as light condition,conidial concentration,nutrient resources and pH value of conidial suspension were studied.There was no significant difference among light treatments.The optimal pH for conidium germination and appressorium formation was 5.0 to 7.0.The exogenous nutrient sources were not the indispensable factors for conidium germination,but 5% sugar solution were more favorable for appressorium formation than the others.Low conidial concentration in suspension (conidia ≤ 10⁴/mL) was propitious to conidium germination and appressorium formation,which were inhibited significantly in higher concentration.It was suggested that the phenomenon was due to the self-inhibitor,a kind of lipophilic substance,existing in the site of conidium germination.     

Use of sclerotia of<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> for efficient production of conidia of<Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in liquid culture

A study was conducted to determine the feasibility of using sclerotia ofSclerotinia sclerotiorum for producing conidia ofConiothyrium minitans in liquid culture. The medium (SST) was made of water containing 2.0, 1.5, 1.0 or 0.5% (w/v) ground sclerotia ofS. sclerotiorum and 100 μgl ⁻¹ thiamine hydrochloride (HCl). One ml of conidial suspension (2 × 10⁷ conidia ml⁻¹) ofC. minitans LRC 2534 was inoculated into 100 ml of SST medium or control (thiamine HCl in water) and incubated at 20 ± 2°C on a shaker at 200 rpm. Subsamples were removed periodically and examined under a compound microscope. Conidia in the SST media germinated within 24 h, developed into branched hyphae within 48 h, produced pycnidia after 3–4 days, and the pycnidia released mature conidia after 7 days. Production of conidia varied with the concentration of sclerotia in the SST medium. It was lower (3.6 × 10⁶ conidia ml⁻¹) at 0.5% but higher (1.2 × 10⁸ conidia ml⁻¹) at 2%. The new conidia were viable and the colonies developing from them showed the original morphological characteristics. It was concluded that using SST liquid medium as a substrate for mass production of conidia ofC. minitans has potential for use in commercial development of this mycoparasite as a biocontrol product. http:www.phytoparasitica.org posting Jan. 23, 2007.     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

Phylogenetic analysis based on ITS sequences and conditions affecting the type of conidial germination of Bipolaris oryzae

One taxonomic characteristic of Bipolaris species is the bipolar germination of conidia, but conidia of Bipolaris oryzae , the causal pathogen of brown spot in rice, are regularly observed to show intercalary germination, a characteristic of Drechslera species. The effect of selection, culture media and culture age on type of conidial germination was determined for three brown spot isolates from Cavinti, San Pablo and Palawan in the Philippines, obtained from infected leaves showing typical disease symptoms. Based on the analyses of their ITS1, ITS2 and 5·8S rDNA nucleotide sequences, the local isolates were clearly identified as B. oryzae . Selection for colonies of the three isolates derived from single conidia with either bipolar or intercalary germination had no effect on the number of spores showing bipolar germination in subsequent cultures. Germination on seven different culture media was tested; of these, rabbit food agar and water agar increased the percentage of bipolar germination of conidia, although this varied between isolates. Incubation of the cultures of all three isolates for longer periods prior to harvesting conidia increased the percentage of bipolar-germinating conidia from c . 40 to c . 90% with 5-day-old and 30-day-old cultures, respectively.     

Non-host resistance toColletotrichum lagenariumin pumpkin and squash is not primarily associated with β-1,3-glucanase and chitinase activities

Disease symptoms typical of anthracnose were observed in cucumber, pumpkin and squash after infiltrating leaves with a conidial suspension ofColletotrichum lagenarium, but symptoms developed only in cucumber when droplets of the conidial suspension were applied to the leaf surface. There was no difference in the germination of conidia or in appressorium formation on leaf surfaces of cucumber, pumpkin or squash plants; however, penetration was markedly reduced into pumpkin and squash with or without systemic acquired resistance (SAR) and into cucumber with SAR. Little β-1,3-glucanase and chitinase activities were detected in challenged pumpkin and squash leaves without symptoms even 5 days after inoculation in leaf surfaces. However, the enzymes were detected in pumpkin and squash leaves with symptoms, and activities of the enzymes were greater than those in cucumber. These results suggest that β-1,3-glucanase and chitinase activities are not primary initial defence compounds associated with non-host resistance of pumpkin and squash toC. lagenarium.     

Suppression of conidial germination and appressorial formation by silicate treatment in powdery mildew of strawberry

The mode of action of soluble silicon against strawberry powdery mildew (Sphaerotheca aphanis var. aphanis) was investigated in four experiments. First, silicon-treated leaves from plants grown with silicate (Si+) and control leaves were excised, inoculated with conidia, and subsequent germination and formation of appressoria in a petri dish was assessed after 24 h. The germination rate was 49.7% on Si+ leaves, and was 67.2% on control leaves (t-test, P < 0.01). Second, we soaked cellulose membranes in various solvents and then placed the membranes on 4% water agar, dusted the membranes with conidia, and examined after 12 h. No difference was apparent between any treatment and the control (distilled water). Third, strawberries growing hydroponically with additional silicon in the medium were inoculated with conidia, and leaves were observed with a scanning electron microscope 1–2 days after inoculation. Germ tubes and secondary hyphae were shorter and had fewer branches on Si+ leaves than on the control. Moreover, penetration appeared to be inhibited. Fourth, the cuticle was separated from leaves from plants grown as in the third experiment, placed on water agar, and dusted with conidia. Germination of conidia, observed with a light microscope, on Si+ leaves was suppressed markedly to 40%–60% of that of the control. These results suggested that soluble silicon induced physiological changes in the cuticle layer after absorption by the plant. In addition, soluble silicate reduced germination of conidia, formation of appressoria, and possibly the penetration of powdery mildew.     

Germination and Sporulation of Colletotrichum acutatum on Symptomless Strawberry Leaves

ABSTRACT The germination and sporulation of Colletotrichum acutatum were characterized over time on strawberry leaves (cv. Tristar) and plastic coverslips incubated at 26 degrees C under continuous wetness. Conidia germinated within 3 h after inoculation and formed melanized appressoria with pores by 9 h after inoculation. Host penetration was not observed up to 7 days after inoculation. Production of secondary conidia on conidial and hyphal phialides began within 6 h after inoculation. Secondary conidiation was responsible for up to a threefold increase in the total number of conidia within 7 days after inoculation. Primary conidia and hyphae began to collapse 48 h after inoculation, whereas melanized appressoria remained intact. These findings suggest that appressoria and secondary conidia of C. acutatum produced on symptomless strawberry foliage may be significant sources of inoculum for fruit infections.