Control of infection and sporulation ofBotrytis cinerea on bean and tomato by saprophytic bacteria and fungi

Sixty isolates of saprophytic microorganisms were screened for their ability to reduce the severity of grey mould (Botrytis cinerea) infection and sporulation. Isolates of the bacteriaXanthomonas maltophilia, Bacillus pumilus, Lactobacillus sp., andPseudomonas sp. and the fungusGliocladium catenulatum reduced germination of conidia of the pathogen and controlled disease on bean and tomato plants. Their activity under growth room conditions was good, consistent, and similar to the activity of the known biocontrol agent,Trichoderma harzianum T39 (non-formulated). Although the tested isolates may for nutrients with the germinating conidia ofB. cinerea, resistance induced in the host by live or dead cells were also found to be involved. Inhibitory compounds were not detected on treated leaves. Sporulation ofB. cinerea after its establishment on leaves was also reduced by the above mentioned isolates and byPenicillium sp.,Arthrinium montagnei, Ar. phaeospermum, Sesquicillium candelabrum, Chaetomium globosum, Alternaria alternata, Ulocladium atrum, andT. viride. These sporulation-inhibiting fungi did not reduce the infection of leaves byB. cinerea. Most of these selected fungi and bacteria were capable of reducing lesion expansion.     

Biological control ofBotrytis cinerea in residues and flowers of Rose (Rosa hybrida)

Microbial isolates from living petals, petal residues and leaf residues of rose, and from laboratory collections, were evaluated for control ofBotrytis cinerea in rose. In leaf residues artificially infested withB. cinerea, isolates of the filamentous fungiGliocladium roseum, FR136 (unidentified) andTrichoderma inhamatum reduced sporulation of the pathogen by >90%, other filamentous fungi were 25–90% effective, and those of yeasts and bacteria were <50% effective. In artificially inoculated petal residues, no microbe reduced sporulation ofB. cinerea by >75%, but isolates ofCladosporium oxysporum and four yeasts were 51–75% effective, and three filamentous fungi, eight yeasts andBacillus subtilis isolates were 26–50% effective. Isolates ofT. inhamatum, C. oxysporum andG. roseum performed best againstB. cinerea among isolates evaluated in leaf residues naturally infested with the pathogen and indigenous microorganisms. Totals of ten isolates of filamentous fungi (includingC. oxysporum andC. cladosporioides), two of yeasts and five ofBacillus subtilis completely prevented lesion production byB. cinerea in detached petals, and a further six isolates of filamentous fungi (includingG. roseum) and six yeasts were 90–99% effective. Isolates ofC. oxysporum, C. cladosporioides andB. subtilis, the most effective microorganisms againstB. cinerea in flower buds, reduced number of lesions in the range of 42–65% compared with 59–89% for à standard fungicide (vinclozolin). It is suggested that application of leading antagonists Jo living rose leaves and flowers should optimize control of inoculum production byB. cinerea when the tissues die. Optimal biocontrol of lesion production in flower buds requires a better understanding of the microenvironment of petals.     

Efficiency of isolates ofConiothyrium minitans as mycoparasites ofSclerotinia sclerotiorum,Sclerotium cepivorum andBotrytis cinerea on tomato stem pieces

Summary Twenty five isolates ofConiothyrium minitans were screened for antagonism toSclerotinia sclerotiorum in a Petri dish bioassay using tomato stem segments placed on sterile sand. The antagonistic activity of 23 isolates was quite uniform and only two less antagonistic isolates were identified. Antagonism, expressed as a reduction in the rate of tissue colonization byS. sclerotiorum, occurred, whetherC. minitans was co-inoculated at the same time, one day before or one day afterS. sclerotiorum, but was slightly restricted whenS. sclerotiorum was given a lead of one day. On average, 50–80% of sclerotia of S.sclerotiorum formed on the stem pieces were infected byC. minitans two weeks after inoculation. Excluding the less antagonistic isolates,Coniothyrium minitans was recovered from over 80% ofS. sclerotiorum-infected stem segments when co-inoculated but from a maximum of only 7% of stem pieces when exposed toC. minitans alone. When the experiments were carried out on non-sterile soil instead of sterile sand, infection of stem pieces byS. sclerotiorum was reduced and recovery ofS. sclerotiorum andC. minitans from stem segments was decreased. SevenC. minitans isolates were also screened againstSclerotium cepivorum andBotrytis cinerea and, whereas the effect ofC. minitans onS. cepivorum-infected tissue and sclerotia was essentially similar to that observed withS. sclerotiorum, B. cinerea infected tissue and sclerotia were not invaded by the antagonist.     

The Role of Trichoderma harzianum Protease in the Biocontrol of Botrytis cinerea

The role of protease of Trichoderma harzianum in the biocontrol of Botrytis cinerea was examined. Two isolates of T. harzianum were compared for their ability to produce protease in liquid culture medium and on the surface of bean leaves. The biocontrol agent T. harziaum T39 produced 58mU/ml of protease and T. harzianum NCIM1185 produced 54mU/ml on the 5th day of growth in liquid culture medium. On bean leaves, combinations of B. cinerea and T. harzianum isolates were examined for the synthesis of protease. The protease activities were 0.9 and 0.6mU/ml for T. harzianum T39 and NCIM1185, respectively, and 0.5mU/ml for B. cinerea alone after 48h of incubation. In the presence of T. harzianum T39 culture liquid containing protease, a 55% reduction in B. cinerea germination and a 80% reduction in the germ tube length were observed after 17h of incubation in vitro. When T. harzianum isolates were added to B. cinerea on bean leaves, increased synthesis of protease was observed (1.0 and 1.2mU/ml for T39 and NCIM1185, respectively). In the presence of T. harzianum NCIM1185 protease, although the rate of germination was reduced, B. cinerea attained 98% germination after 17h of incubation. The hydrolytic enzymes produced by B. cinerea, endo-polygalacturonase (PG) and exoPG were partially deactivated by protease from the T. harzianum isolates. Carboxymethyl cellulase was deactivated only by protease of NCIM1185. On the surface of bean leaves, the protease (obtained from liquid culture medium of T. harzianum isolates) resulted in 56–100% reduction of disease severity. The culture liquid containing protease synthesized on the surface of bean leaves treated with B. cinerea and with T. harzianum was collected and added to fresh leaves infected by B. cinerea. There was 56–100% and 30–75% reduction of disease severity with liquid droplet collected from the leaves treated with T. harzianum T39 and NCIM1185, respectively. Increased control of disease was obtained by combining the conidia of T. harzianum isolates with protease obtained from culture media. Protease inhibitors, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), antipain hydrochloride, and a mixture of inhibitors, but not pepstatin A, fully or partially nullified the biocontrol effect of T39. T39 was found to be a poor producer of chitinase and -1,3-glucanase in vitro. These enzymes were not detected on leaves treated with T39. Involvement of protease in biocontrol of B. cinerea is suggested.     

Relationships of aphid and mite infestations to control ofBotrytis cinerea byClonostachys rosea in rose(Rosa hybrida) leaves

Infestations of aphids(Macrosiphum rosae L.) and of twospotted spider mites(Tetranychus urticae Koch) were examined in relation to growth and sporulation ofClonostachys rosea andBotrytis cinerea, and to suppression of the pathogen by the agent, in green rose leaves. Leaves were infested artificially with 10 aphids/leaflet for 3 h, or naturally with 15-30 aphids/leaflet for 7-12 days or with undetermined numbers of mites for 10-12 days. Leaves that had or had not been infested were inoculated withC. rosea, withB. cinerea, or withC. rosea plusB. cinerea. Germination incidence and germ tube growth ofC. rosea andB. cinerea on the phylloplane in most instances were much greater in leaves previously infested with aphids or mites compared with noninfested leaves. After combined inoculation,C. rosea suppressed germination ofB. cinerea from 47% to 19% in noninfested leaves, but in leaves that had been infested the agent was ineffective and germination incidence of the pathogen increased to 75-93%. Previous infestation with naturally introduced aphids or mites, but not brief infestations of artificially introduced aphids, markedly increased sporulation ofC. rosea after the leaves died during an initial 7-15 days of incubation on a paraquat agar medium, regardless of whether or notB. cinerea was present. Sporulation ofB. cinerea was similarly increased when inoculated alone. After 15-20 days, however, conidiophores of the agent or pathogen covered most of the leaf surface in these treatments. In leaves inoculated withC. rosea plusB. cinerea, the agent suppressed sporulation of the pathogen almost completely in both previously infested and noninfested leaves. Thus, aphid and mite infestations did not compromise the ability ofC. rosea to suppress inoculum production byB. cinerea in the leaves. Increased nutrient availability on the phylloplane through exudation or as honeydew or frass is proposed as a basis to explain effects of the pest infestations onC. rosea andB. cinerea.     

芽胞杆菌JZB30-1的鉴定及其对番茄灰霉病的生防作用

为获得对番茄灰霉病具有良好防效的生防芽胞杆菌, 采用对峙培养、双层平板以及生防相关特性检测等方法筛选高活性拮抗菌株, 根据形态学、生理生化特性及多基因测序进行菌株种类鉴定, 通过离体果实和盆栽试验明确供试菌株的防病促生效果。结果表明, 芽胞杆菌JZB30-1对番茄灰霉病菌等10余种植物病原真菌和细菌具有广谱抑菌活性; 该菌株具有合成蛋白酶、纤维素酶、脂肽类物质,分泌铁载体、生长激素及溶磷等特性, 经鉴定其为解淀粉芽胞杆菌Bacillus amyloliquefaciens; 菌株JZB30-1无菌发酵滤液50倍液喷施处理, 对番茄果实灰霉病防效为95.0%, 发酵液10倍稀释液灌根, 番茄株高、鲜重分别较对照增加25.7%和28.4%。相关结果为利用解淀粉芽胞杆菌JZB30-1进行番茄灰霉病生物防治提供理论依据。     

Honey bee dispersal of the biocontrol agent Trichoderma harzianum T39: effectiveness in suppressing Botrytis cinerea on strawberry under field conditions

Botrytis cinerea, which causes grey mould, is a major pathogen of many crops. On strawberry, isolates of Trichoderma spp. can effectively control B. cinerea, but frequent application is necessary. Bees can be used to disseminate biological control agents to the target crop. We tested the ability of honey bees to disseminate Trichoderma harzianum T39 to control B. cinerea in strawberry in the field during the winter in Israel over two consecutive seasons. We used the recently developed ‘Triwaks’ dispenser for loading the bees with the T. harzianum inoculum. During both years, grey mould developed in late January in untreated control plots; at low to medium disease levels it was partially controlled by fungicide treatment, and was best controlled in bee-visited plots. At high disease levels neither chemical nor biological control was effective. To assess the spatial distribution of inoculum by bees, we sampled flowers up to 200 m from the hives and found effective levels of T. harzianum even at 200 m. The approach used in this study provides an effective control of grey mould in strawberry in conditions of low to medium grey mould incidence.     

Comparison of Antagonists of Botrytis cinerea in Greenhouse-grown Cucumber and Tomato under Different Climatic Conditions

The efficacy of Trichoderma harzianum T39 and the yeasts Aureobasidium pullulans and Cryptococcus albidus against Botrytis cinerea in cucumber and tomato was compared with chemical control. Four experiments were conducted in cucumber grown under different climatic conditions in The Netherlands, and two experiments were done in tomato both in the Netherlands and in Israel. T. harzianum and A. pullulans showed the most consistent control of B. cinerea, reducing stem lesions and death of plants by 40–100% in most cases. Control of stem lesions and subsequent wilting was generally better than control of symptoms on fruits. In some cases, the biocontrol agents were more effective than the broad-spectrum fungicide tolylfluanid and the selective fungicide iprodione. The climatic conditions did not strongly influence the efficacy of the biocontrol agents, but regression analysis showed that high temperature during the day and high vapour pressure deficit during the night reduced biocontrol efficacy. From the results, prospects for biocontrol of B. cinerea in greenhouse vegetables appear good under a range of conditions.     

Induced systemic resistance in Trichoderma harzianum T39 biocontrol of Botrytis cinerea

Biocontrol of Botrytis cinerea with Trichoderma spp. is generally believed to result from direct interaction of the biocontrol agent with the pathogen or from a Trichoderma-induced change in environmental conditions that affects B. cinerea development. In this work we provide arguments for the participation of induced plant defence in T. harzianum T39 control of B. cinerea. In tomato, lettuce, pepper, bean and tobacco, T. harzianum T39 application at sites spatially separated from the B. cinerea inoculation resulted in a 25–100%percnt; reduction of grey mould symptoms, caused by a delay or suppression of spreading lesion formation. Given the spatial separation of both micro-organisms, this effect was attributed to the induction of systemic resistance by T. harzianum T39. The observation that in bean the effect of T. harzianum T39 was similar to that of the rhizobacterium Pseudomonas aeruginosa KMPCH, a reference strain for the induction of systemic resistance, confirmed this hypothesis. Since B. cinerea control on tobacco leaves sprayed with T. harzianum T39 was similar to the control on leaves from T. harzianum T39 soil-treated plants, induction of plant defence might also participate in biocontrol on treated leaves.     

哈茨木霉菌与5种杀菌剂对番茄灰霉病菌的协同作用

采用对峙法、菌丝生长速率法和离体叶片法分别测定了哈茨木霉菌Trichoderma harzianum与5种不同作用机制杀菌剂联用对番茄灰霉病菌Botrytis cinerea的抑制作用。结果表明,联用可增强哈茨木霉菌及啶酰菌胺、嘧菌酯、咯菌腈、氟啶胺和啶菌噁唑5种供试药剂对番茄灰霉病菌的抑制作用,即具有协同作用,因此可考虑将杀菌剂与木霉菌联合用于番茄灰霉病的防治。     

Effect of interrupted leaf wetness periods on suppression of sporulation ofBotrytis allii andB. cinerea by antagonists on dead onion leaves

Saprophytic antagonists were evaluated for suppression of sporulation ofBotrytis allii andB. cinerea on artificially killed segments of onion leaves that were pre-inoculated with the pathogens. During incubation of the antagonisttreated leaf segments in moist chambers, periods of leaf wetness and leaf dryness were alternated to simulate conditions in the field. Interruption of humid conditions with dry periods had a differential effect on antagonists.Alternaria alternata, Chaetomium globosum, Ulocladium atrum andU. chartarum suppressed sporulation ofB. allii almost completely under continuously wet conditions, and when the leaf wetness periods were interrupted with drying periods of 9h imposed 16, 40, and 64 h after the antagonists were applied. When leaf wetness was interrupted 16 h after antagonist application, the number of conidia ofB. allii produced cm^–2 leaf surface after eight days was under the detection limit of 5.2 × 10³ conidia on leaves treated with these antagonists compared to 3.7 × 10⁵ conidia on leaves that were not treated. On the other hand,Gliocladium roseum, G. catenulatum andSesquicillium candelabrum, all highly efficient under continuously wet conditions, were of low to moderate efficiency when leaf wetness periods had been interrupted 16 h after application of the antagonists. The antagonists showed the same differentiation and sensitivity to interrupted wetness periods when tested withB. cinerea.     

哈茨木霉Th-30几丁质酶的生产条件及对灰霉病菌的拮抗作用

病菌生长的抑制率为67.61%～71.31%,对灰霉病菌孢子产生及萌发的抑制率分别为80.95%和81.35%,对病菌孢子致病性的抑制率为65.22%～70.51%.     

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。     

Combined effect of generally regarded as safe (GRAS) compounds andTrichoderma harzianum on the control of postharvest diseases of rambutan

Botryodiplodia theobromae, Colletotrichum gloeosporioides andGliocephalotrichum microchlamydosporum are the causal fungi of the rambutan postharvest diseases stem-end rot, anthracnose and brown spot, respectively. Two different treatments of rambutan fruits(Nephelium lappaceum) against the three pathogens were compared: potassium metabisulphite (250 ppm) or cinnamaldehyde (30 ppm), each combined withTrichoderma harzianum (TrH 40). The application of TrH 40 and potassium metabisulphite effectively controlled the incidence and severity of the three postharvest diseases and maintained the overall quality and color of the fruit under low temperature storage at 13.5°C and 95% r.h. for 18 days. The greatest effect of this treatment was shown onG. microchlamydosporum. Cinnamaldehyde affected the growth and germination of TrH 40, whereas potassium metabisulphite did not. http://www.phytoparasitica.org posting Nov. 4, 2001.     

复合微生物菌剂对苹果再植病害的生防效果测定及拮抗菌株鉴定

为探究复合微生物菌剂对苹果再植病害的生防效果,以苹果砧木实生海棠幼苗为试材,通过盆栽试验测试了木美土里复合微生物菌剂对苹果再植病害的防治效果及对再植海棠的促生作用,然后对该菌剂的可分离微生物进行了分离纯化,并以尖孢镰刀菌Fusarium oxysporum菌株HS2为靶标菌,测试分离获得的10株菌株的拮抗效果,并对拮抗效果显著的1株真菌菌株和2株细菌菌株进行种类鉴定。结果表明,木美土里复合微生物菌剂对苹果再植病害的防治效果达87.78%;经该菌剂处理后,再植海棠苗的株高、茎粗、鲜重、干重及叶面积分别是对照处理的1.51、2.00、5.74、5.21和2.83倍,表明该菌剂有显著的促生作用。木美土里复合微生物菌剂对菌株HS2的抑制率达到68.59%,并从中分离到7株真菌菌株,3株细菌菌株。其中,拮抗效果高于80.00%的真菌菌株有1株,细菌菌株有2株。经鉴定,真菌菌株MMTL-1为哈茨木霉Trichoderma harzianum;细菌菌株BM-01和BM-03均为解淀粉芽胞杆菌Bacillus amyloliquefaciens。     

Evaluation of Microbial Antagonists for Biological Control of Botrytis cinerea Stem Infection in Cucumber and Tomato

Of fifteen isolates of yeasts, filamentous fungi and bacteria and a commercial product, tested in a bioassay with stem segments, eleven isolates consistently reduced incidence of disease and sporulation of Botrytis cinerea Pers; Fr in tomato and seven isolates in cucumber. Several isolates reduced disease by more than 75% in all experiments. Six antagonists that performed well in the bioassays and that were fairly easy to produce in vitro, were selected for further testing in two glasshouse experiments with cucumbers. After application of spores of B. cinerea and the antagonists or the fungicide tolylfluanid to pruning wounds, disease incidence was reduced by 50–100% by all antagonists in both experiments and only in one experiment by tolylfluanid.For Trichoderma harzianum T39, Aureobasidium pullulans and Cryptococcus albidus, biological control efficacy in bioassays with cucumber stem segments was not strongly influenced by temperatures in the range between 18 and 30°C, but at 24°C the efficacy of the three antagonists strongly decreased at relative humidities of 90% and 80% (vapour pressure deficits 0.299 and 0.598kPa, respectively) compared to 100.     

Antagonistic effect ofTrichoderma harzianum on postharvest pathogens of rambutan (Nephelium lappaceum)

Trichoderma harzianum (TrH 40) isolated from soil samples from rambutan orchards (Nephelium lappaceum) had antagonistic effects against three postharvest pathogens of rambutan:Botryodiplodia theobromae, Colletotrichum gloeosporioides andGliocephalotrichum microchlamydosporum, causative fungi of stem end rot, anthracnose and brown spot, respectively. The effects were due to both antibiosis and mycoparasitism.T. harzianum (TrH 40) treatment, while reducing the occurrence of the three postharvest diseases, also retained the overall quality and color of the fruits.     

番茄茎基腐病病原菌的生物学特性

对番茄茎基腐病病原菌生物学特性研究结果表明:病菌菌丝在供试PDA、PSA、2%水琼脂、Richard、番茄煎汁、燕麦片和番茄燕麦等7种培养基中均能良好生长,而在2%水琼脂培养基上生长较为缓慢;菌丝在2～32℃范围内均能生长,最适20℃,致死温度为50℃,10min;菌丝生长的pH范围为3～10,最适为5～7;光照对菌丝生长影响较小。病菌在7种供试培养基上均能产生菌核;菌核在10种供试营养物质中均能萌发,萌发温度范围为5～30℃,最适20℃,致死温度为59℃,10min;菌核萌发pH范围为3～10,最适为7。     

Evaluation of biocontrol preparations and plant extracts for the control of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> on potato leaves

The potential of biocontrol products and plant extracts for control of late blight on potato plants, caused by Phytophthora infestans was evaluated in detached leaf assays and on potted plants. Based on an initial screening of 22 preparations and plant extracts, the 10 most active treatments were selected for further investigation. In the detached leaf assays the commercial preparations Elot-Vis, Serenade and Trichodex, and plant extracts of Rheum rhabarbarum and Solidago canadensis showed a significant effect on the level of infestation by P. infestans. However, none of the treatments was as effective as copper. In the case of Serenade, the metabolites produced by its active micro-organism, Bacillus subtilis, were demonstrated to be the effective component of the formulation, and not the micro-organism itself. In order to take curative and protective modes of action into account, the test substances were applied 24 h before, or 90 min after inoculation with P. infestans. Generally, better effects were obtained when the applications were made 24 h before inoculation. For defining the optimum time of application, potted plants were treated 72 or 24 h before, and 1 and 24 h after inoculation with P. infestans. In these tests, Trichodex showed no activity, while Elot-Vis gave best results when applied 1 day before inoculation. Serenade and the extracts of R. rhabarbarum and S. canadensis (all at 5% concentration) however, were effective when applied up to 3 days before and just after inoculation with P. infestans. The results of the experiments on potted plants indicated direct effects on the pathogen for all agents except the extract of S. canadensis, but other mode of actions, e.g. induced resistance, could not be ruled out. None of the treatments had a curative effect.     

石榴皮萃取物对番茄灰霉病菌抑制作用及防病效果

试验测定了石榴皮萃取物对番茄灰霉病菌室内抑菌作用和对番茄灰霉病的温室盆栽防治作用。室内抑菌试验分别采用生长速率法和孢子萌发法测定对番茄灰霉病菌菌丝生长和孢子萌发的抑制作用,结果表明：正丁醇萃取物对病原菌菌丝的抑制活性最高,EC50为50.28 g/L;浓度为200 g/L对番茄灰霉病菌分生孢子的产生具有显著的抑制作用,抑制作用与浓度呈正相关关系。在温室盆栽试验中, 正丁醇萃取物稀释100倍的处理对番茄灰霉病的保护防治效果达到46.41%,治疗防治效果达到48.00%。