Pathogenicity of the fungus, Verticillium lecanii, to the green peach aphid, Myzus persicae (Hom.: Aphididae)

Pathogenicity of the hyphomycete, Verticillium lecanii (DAOM 198499), was investigated to aphid, Myzus persicae under laboratory conditions. Analysis of lethal effect of six various concentrations 0, 10(4), 10(5), 10(6), 10(7) and 10(8) conidia/ml of V. lecanii against third nymphal stage of M. persicae, indicated significant mortality on aphids. Mean comparisons showed that there was significant difference between treatments. Three days after treatment, aphid mortality observed and after 12 days minimal mortality was 17.77 in control and maximum was 100 percent related to 10(7) and 10(8) conidia/ml. LC50 and LT50 values were estimated by probit analysis and life test. LC50 value for aphid mortality was 1.4 x 10(4) conidia/ml, LT50 values for concentrations 10(4), 10(6), 10(6), 10(7) and 10(8) conidla/ml was 10, 10, 9, 8 and 6 days, respectively. In this experiment, net reproduction rate of aphid's (R0) decreased significantly when concentration increased. These observations showed that V. lecanii (DAOM 198499) can be an active biological agent against aphids.     

Evaluation of Lecanicillium longisporum, Vertalec for simultaneous suppression of cotton aphid, Aphis gossypii, and cucumber powdery mildew, Sphaerotheca fuliginea, on potted cucumbers

The commercial preparation of Lecanicillium longisporum, Vertalec^® was evaluated for simultaneous suppression of cotton aphid and cucumber powdery mildew on potted cucumber plants. Vertalec was applied onto cucumber plants that had been infested with either cotton aphid, spores of Sphaerotheca fuliginea or both. Irradiation-inactivated Vertalec (II Vertalec) was also applied to an identical series of cucumber plants as a control. The Vertalec was highly pathogenic against adult aphids with an LT₅₀ of 6.9 days. II Vertalec did not affect aphid survival. Application of either active or II Vertalec significantly suppressed spore production of S. fuliginea compared to the water control. For dual control assays, Vertalec applications were made one day after infestation of both aphid and S. fuliginea onto potted cucumbers. Fifteen days after the Vertalec treatments, the numbers of surviving aphids and the production of powdery mildew spores were significantly reduced compared with the water control. The presence of aphids also suppressed S. fuliginea spore production. Our results suggest the potential of a dual role for Vertalec as a microbial control agent of aphids and powdery mildew in cucumber.     

Preliminary evaluation of the effectiveness of a Verticillium lecanii isolate in the control of Thrips tabaci (Thysanoptera: Thripidae)

Thrips tabaci is a polyphagous pest that attacks many different crop and ornamental plant species. Recently, the need to reduce the use of insecticides in the control of thrips is more and more increasing. On that account, the possibility to make use of the entomopathogenic fungus Verticillium lecanii (DAOM198499) was evaluated in laboratory conditions. Suspension of 10(3) to 10(7) conidia/ml grown in liquid culture medium with a control (distilled water) treated on second larval stage of T. tabaci in controlled condition (16L: 8D photoperiod, 25 +/- 1 degrees C temperature, 98% RH in first 48h and 70% RH in next days). Preliminary results showed that mortality in lethal suspension (10(7) conidia/ml) of V. lecanii after 24 hours and 2-7 days in lower suspension appears. This suggests that strine DAOM 198499 of V. lecanii has the ability to control T. tabaci in greenhouse conditions.     

Potential of Lecanicillium species for dual microbial control of aphids and the cucumber powdery mildew fungus, Sphaerotheca fuliginea

Detached leaf disc bioassays were conducted against cucumber powdery mildew and three species of aphid with three entomopathogenic species of Lecanicillium; Lecanicillium longisporum (Vertalec^®), Lecanicillium attenuatum (CS625), and an unidentified isolate (DAOM198499). The three Lecanicillium species had high virulence against the aphids Myzus persicae, Macrosiphum euphorbiae and Aulacorthum solani with the exception of DAOM 198499, which demonstrated reduced virulence to A. solani with an LT₅₀ of 6.4 days. Otherwise, LT₅₀ ranged between two and four days. Suspensions of conidia and blastospores of the Lecanicillium species were also applied onto 15 mm leaf discs dissected from cucumber plants previously inoculated with Sphaerotheca fuliginea. Powdery mildew did not develop when the Lecanicillium applications were made one and eight days after S. fuliginea inoculations. When Lecanicillium was applied to highly infected leaf discs 11 and 15 days after S. fuliginea inoculation, the application suppressed subsequent production of S. fuliginea spores as compared to the controls. These results suggest the potential of a dual role for Lecanicillium spp. as microbial control agents against aphids and powdery mildew.     

Improvement of the efficacy of Verticillium lecanii used in biocontrol of Sphaerotheca fuliginea by addition of oil formulations

Biocontrol of cucumber powdery mildew, Sphaerotheca fuliginea, by Verticillium lecanii is seriously hampered at low humidities. The effect is especially marked at low humidity (60% RH) during the three hours following the application of V. lecanii spores suspended in water. Formulations of V. lecanii spores in oil might improve the situation. Arachid oil (peanut oil) and two invert emulsions using either Sunspray 6N or paraffin oil were tested in formulations of V. lecanii spores. Arachid oil gave the best development of V. lecanii on mildewed cucumber leaves. V. lecanii formulated with arachid oil showed significantly better control of mildew than without. A concentration of 0.5% arachid oil was somewhat toxic to mildew but 0.05% was not. Arachid oil did not show toxicity to V. lecanii. The humidity requirements of V. lecanii formulated with and without 0.05% arachid oil were compared at 95, 90 and 85% RH. Arachid oil significantly reduced the humidity dependence of V. lecanii. Since arachid oil is safe for human consumption and not phytotoxic to cucumber leaves, low concentrations of arachid oil are recommended as an additive to increase the effectiveness of V. lecanii as a biocontrol agent of S. fuliginea.     

Preventative and Curative Applications of Verticillium lecanii for Biological Control of Cucumber Powdery Mildew

The effect of timing of the application of the mycoparasite , Verticillium lecanii, on cucumber powdery mildew , Sphaerotheca fuliginea, was studied in a rooted cucumber leaf bioassay . The mycoparasite was applied at different times before and after mildew inoculation . At near - to maximum humidity ( 95% relative humidity) , early preventative (9 and 5 days before mildew inoculation) and early curative control treatments (2 days after mildew inoculation) gave considerable reduction in mildewed leaf areas , while late curative treatments resulted in greater mildewed leaf area but ultimately a reduced amount of healthy mildewed leaf area ( 20 % ) . Appropriate timing of biocontrol treatment application of V. lecanii is important to achieve good control .     

Comparison of laboratory infection of aphids by Metarrhizium anisopliae and Verticillium lecanii

The pathogenicity of the only documented ‘aphid’ strain of Metarrhizium anisopliae for the aphid, Macrosiphoniella sanborni, was established by experimental infection. Its aphicidal potential was assessed by conducting parallel experiments with Verticillium lecanii, a fungus of proven efficacy in controlling aphids. The spore germination, growth and sporulation rates of M. anisopliae were slower than V. lecanii and were most likely the principal factors determining the higher LC., values in bioassay and slower propagation of this fungus amongst aphid populations. It is concluded that M. anisopliae, except perhaps in the salt marsh habitat from which it was originally isolated, would not prove as useful an agent as V. lecanii in biological control of aphids.     

Selection of Verticillium lecanii Isolates with High Potential for Biocontrol of Cucumber Powdery Mildew by Means of Components Analysis at Different Humidity Regimes

To identify characteristics for the selection of Verticillium lecanii isolates with high potential for biocontrol of Sphaerotheca fuliginea under glasshouse conditions, an exploratory study was performed on the effect of water limitation on the development of 14 isolates. The conidial germination, growth and sporulation of isolates of V. lecanii were studied in a tritrophic system on cucumber leaves and in a ditrophic system in Petri dishes. Their mycoparasitic ability was studied in S. fuliginea and Cladosporium cladosporioides . All characteristics were clearly affected by humidity. Four isolates showed good biocontrol potential. The performance of isolates on agar had less predictive value than on powdery mildew. The germination of isolates of V. lecanii was lower and the mycelial growth faster on agar than on mildewed leaves under corresponding humidity conditions. The results suggest that conditions in the phyllosphere differed from the set humidity in the surrounding air. A correlation was found between the lysis of C. cladosporioides growing in dual culture on agar with isolates of V. lecanii and the parasitism of powdery mildew on detached, rooted leaves. C. cladosporioides might offer a suitable substrate for testing isolates of V. lecanii for mycoparasitic potential under various environmental conditions. Conidial germination, growth and sporulation had limited predictive value.     

Indirect Effect of a Transgenic Wheat on Aphids through Enhanced Powdery Mildew Resistance

In agricultural ecosystems, arthropod herbivores and fungal pathogens are likely to colonise the same plant and may therefore affect each other directly or indirectly. The fungus that causes powdery mildew (Blumeria graminis tritici) and cereal aphids are important pests of wheat but interactions between them have seldom been investigated. We studied the effects of powdery mildew of wheat on two cereal aphid species, Metopolophium dirhodum and Rhopalosiphum padi. We hypothesized that aphid number and size will be smaller on powdery mildew-infected plants than on non-infected plants. In a first experiment we used six commercially available wheat varieties whereas in the second experiment we used a genetically modified (GM) mildew-resistant wheat line and its non-transgenic sister line. Because the two lines differed only in the presence of the transgene and in powdery mildew resistance, experiment 2 avoided the confounding effect of variety. In both experiments, the number of M. dirhodum but not of R. padi was reduced by powdery mildew infection. Transgenic mildew-resistant lines therefore harboured bigger aphid populations than the non-transgenic lines. For both aphid species individual size was mostly influenced by aphid number. Our results indicate that plants that are protected from a particular pest (powdery mildew) became more favourable for another pest (aphids).     

The phytopathogen powdery mildew affects food-searching behavior and survival of <Emphasis Type="Italic">Coccinella septempunctata</Emphasis>

The diet of entomophagous coccinellids is mainly based on aphids and other food sources such as pollen, nectar, or fungal spores. Knowledge of their foraging behavior on plants infected by powdery mildew and their survival on fungal spores is currently limited. In this study, we investigated the olfactory response of Coccinella septempunctata to odor emission of barley plants infected by powdery mildew and their survival on fungal spores in the presence or absence of aphids. Odors released by powdery-mildew infected plants were more attractive for ladybirds compared to those of uninfected controls. After 3 days, the survival rate of ladybirds feeding only on powdery-mildew spores was less than 50%, while for ladybirds feeding exclusively on Rhopalosiphum padi aphids, the survival rate was close to 90%. After 15 days, the highest survival rate (almost 80%) was observed for ladybirds feeding on plants with both aphids and powdery mildew. Molecular analyses confirmed the presence of fungal spores in ladybird guts when feeding either on powdery mildew or on a mixed diet. Our results provide new insights into foraging behavior of entomophagous coccinellids revealing the potential of powdery mildew to be utilized as important non-essential food in a mixed diet, but also its lethal effect if consumed alone.     

Glasshouse Experiments on Biocontrol of Cucumber Powdery Mildew (Sphaerotheca fuliginea) by the MycoparasitesVerticillium lecaniiandSporothrix rugulosa

Two potential biological control agents of cucumber powdery mildew (Sphaerotheca fuliginea),Verticillium lecaniiandSporothrix rugulosa,were tested under glasshouse conditions. Two experiments were carried out. In the first experiment, two cucumber varieties with different levels of resistance, cv Corona (susceptible) and cv Flamingo (partially resistant), were used.Verticillium lecaniicontrolled the mildew better thanS. rugulosa.On cv Flamingo,V. lecaniicould keep the mildew severity below 15% infected leaf area for 9 weeks after inoculation withS. fuliginea.Treatment by Hora Oleo 11E, alone or as an additive toV. lecanii,was as good as a fungicide treatment. In the second experiment, weekly and biweekly treatments withV. lecaniiwere compared on cv Flamingo. Weekly treatments withV. lecaniikept mildew severity at a level below 20% infected leaf area during 10 weeks after inoculation withS. fuliginea.If applied to a partially resistant cucumber cultivar,V. lecaniiis an effective candidate for biological control ofS. fuliginea.     

Oleozon: A novel control strategy against powdery mildew in cucumber

Ozonized sunflower oil (oleozon) is an effective agent for controlling powdery mildew in cucumber. In this study, the mechanisms of oleozon in the control of powdery mildew were determined. The development of Podosphaera xanthii on cucumber leaves treated with oleozon (2%) and water was investigated at different times after inoculation. The germinating conidia, hyphae and conidiophores of the pathogen were severely damaged by oleozon. No visible phytotoxic effect was observed on cucumber after the application of oleozon. This compound had highly preventive effects as well as curative effects against powdery mildew based on in vivo potted seedling assays. The control effects of oleozon were further confirmed in a greenhouse trial. These results may provide a basis for further development of a natural fungicide against cucumber powdery mildew.     

Management of powdery mildew and gray mold of cucumber by Trichoderma harzianum T39 and Ampelomyces quisqualis AQ10

Two biocontrol preparations were tested for their ability to control Sphaerotheca fusca and Botrytis cinerea on greenhouse cucumber. Trichoderma harzianum T39 (TRICHODEX) spray reduced powdery mildew severity by up to 97% but its efficacy declined to 18–55% control as the epidemic progressed. Unlike on young leaves, on older leaves the control of powdery mildew by T. harzianum T39 was poor. Ampelomyces quisqualis (AQ10) was very effective against powdery mildew, achieving up to 98% of control. Its effectiveness declined with the progress of the epidemic but unlike the other biocontrol agent it retained significant control capability on older leaves. Two aliphatic petroleum distillate oil products improved the efficacy of both biocontrol agents. The co-application of T. harzianum T39 and A. quisqualis AQ10 was tested on cucumber plants infected with powdery mildew followed by fruit gray mold infection. It resulted in no improvement of the control of powdery mildew, and in an improvement of gray mold control, the latter probably because of the use of additive oil (ADDQ) along with the second biocontrol preparation. There was no significant interference between the biocontrol agents in the co-application treatment as compared with the application of each agent alone; the level of population of T. harzianum T39 remained similar and the parasitism of S. fusca by A. quisqualis was not nullified. The application of T. harzianum T39 to soil instead of spraying it resulted in 75–90% lower powdery mildew coverage on the leaves. It was concluded that the mode of action of T. harzianum T39 in powdery mildew control is induced resistance, not mycoparasitism or antibiotic action.     

亚精胺诱导黄瓜幼苗对白粉病抗性的研究

以黄瓜品种‘长春密刺’幼苗为材料,研究了亚精氨(Spd)诱导黄瓜幼苗对白粉病的抗性,并测定Spd处理和白粉菌接种对黄瓜叶片4种防御酶活性及3种防卫基因表达的影响。结果显示:(1)0.2～1.0mmol.L-1 Spd对黄瓜幼苗白粉病抗性均有不同程度的诱抗效果,并以0.8mmol.L-1 Spd处理效果最明显,诱导效率可达55.3%。(2)喷施Spd或接种白粉菌均可提高黄瓜叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶和β-1,3-葡聚糖酶的活性,且诱导并接种处理的植株叶片上述酶活性均比只诱导不接种处理的上升速度更快;同时,Spd处理和接种白粉菌可以提高植株叶片中POX、PAL、PR-1a基因的表达量。研究表明,Spd处理可以诱导防卫基因表达的增强,提高防御酶活性,显著降低病情指数,增强黄瓜幼苗对白粉病的抗性。     

Antifungal activities of Bacillus thuringiensis isolates on barley and cucumber powdery mildews

Fourteen Bacillus thuringiensis isolates having both insecticidal activity and in vitro antifungal activity were selected and tested for in vivo antifungal activity against tomato late blight, wheat leaf rust, tomato gray mold, and barley powdery mildew in growth chambers. All the isolates represented more than 70% disease control efficacy against at least one of four plant diseases. Specifically, 12 isolates exhibited strong control activity against barley powdery mildew. Under glasshouse conditions, four (50-02, 52-08, 52-16, and 52- 18) of the isolates also displayed potent control efficacy against cucumber powdery mildew. To our knowledge, this is the first report of B. thuringiensis isolates that have disease control efficacy against powdery mildew of barley and cucumber as well as insecticidal activity.     

Characterization of Spanish strains of Verticillium lecanii

We have characterized biologically and physiologically eight Verticillium lecanii strains from several origins including insect pests. Of all the temperatures tested, 25 degrees C was the best for growth and at 40 degrees C none of the strains could grow. At 4 and 7 degrees C, growth was reduced in comparison to warmer temperatures. The strains had better development at pH close to 7 (F = 27.64, P < 0,01) than at pH 3. Self-inhibition of germination of strain 50 was found when more than 0.78 conidia/cm(2) were plated on corn meal agar (CMA). Germination of conidia was close to 100% for all strains except one, three days after inoculation. Among extracellular enzymatic activities studied the fungal strains showed strongest proteolytic activities followed by lipolytic and chitinolytic activities. Some strains showed significant differences (P < 0.05) in conidia production. Most of the fungicides tested (especially benomyl) inhibited radial growth of strain 50 on CMA. Pathogenicity (as median lethal time, LT50) of V. lecanii strains on larvae of Galleria mellonella varied from 2.66 -/+ 0.33 to 4.27 -/+ 0.25 days. We conclude that in vitro tests per se are not sufficient to select the best biocontrol strains of entomopathogenic fungi. Pathogenicity is a complex process in which the presence, timing and regulation of many factors including those covered in this paper, as well as their interactions, are probably involved.     

Powdery mildew susceptibility and biotrophic infection strategies

Plants are resistant to most potentially pathogenic microbes. This forces plant pathogens to develop sophisticated strategies to overcome basic plant resistance, either by masking intrusion or by suppression of host defences. This is particularly true for fungal pathogens, which establish long lasting interactions with living host tissue, without causing visible damage to invaded cells. The interactions of cereal crops and Arabidopsis with powdery mildew fungi are model systems for understanding host resistance. Currently, these systems are also promoting the understanding of fungal infection by identifying fungal pathogenicity and virulence factors and host target sites. This minireview focuses on recent findings about host susceptibility and the way powdery mildew fungi might induce it.     

Pathogenicity of the Hyphomycete Fungi Verticillium lecanii and Metarhizium anisopliae to the Western Flower Thrips,Frankliniella occidentalis

The pathogenicity of several isolates of the hyphomycete fungi Verticillium lecanii and Metarhizium anisopliae to Frankliniella occidentalis was investigated. Treatment of adult thrips with M. anisopliae resulted in at least 94% mortality at 7 days post-inoculation. In contrast, V. lecanii isolates only gave mortalities of between 20 and 70%. Detailed studies were made on the most virulent isolate of M. anisopliae (275) to determine its efficacy at different doses and temperatures. At 23 C the LC was ca. 3 105 conidia ml-1 after 5 days and the LTs were 50 50 3 and 4.5 days at 10 7 and 106 conidia ml-1 respectively. Temperature influenced fungal virulence to adult thrips; the LT at 18 and 20 C was ca. 4 days and at 23 or 26 C it was ca. 3 days. 50 Larvae were less susceptible to infection than adult thrips (27% versus 100% mortality), presumably due to the inoculum being shed with the exuvium during ecdysis. Conidia of M. anisopliae isolate 275 germinated rapidly on the surfaces of larvae, pupae and adults, with most germlings producing appressoria within 24 h post-inoculation. Fungal elements were present in significant amounts in the body 3 days after treatment.     

QTL analysis of powdery mildew resistance in cucumber (Cucumis sativus L.)

A population of F₇ recombinant inbred lines (RILs) was made from a cross between susceptible (‘Santou’) and resistant (PI197088-1) lines of cucumber in order to study powdery mildew resistance loci. Susceptibility to powdery mildew in the F₇ RIL individuals showed a continuous distribution from susceptible to resistant, suggesting that powdery mildew resistance is controlled by quantitative trait loci (QTLs). A QTL analysis identified two and three loci for powdery mildew resistance under 26 and 20°C conditions, respectively. One QTL was found in the same position under both temperature conditions. Therefore, it is more likely that one major QTL acts under both temperature conditions and that other QTLs are specific to the two temperature conditions. The above results suggest that the four QTLs are controlled in a different temperature manner, and that their combination played an important role in expressing a high level of resistance to powdery mildew in this cucumber population. Sequence-tagged site (STS) markers associated with each QTL were developed and would be useful for breeding a cucumber line with a high level of powdery mildew resistance. Y. Sakata and N. Kubo contributed equally to this work and are considered as first authors.     

Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1

Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15–C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.