Penicillium frequentans population dynamics on peach fruits after its applications against brown rot in orchards

Aims: The study provides useful information on the temporal population dynamics of the biological control agent, Penicillium frequentans, after its applications against brown rot in orchards. Methods and Results: Population dynamics of P. frequentans were studied on peach flower and fruit surfaces after different field treatments. Eight experiments were carried out in commercial peach orchards in Spain, over four growing seasons from 2002 to 2005. Six different formulated P. frequentans conidia were applied four to six times from blossom to harvest and P. frequentans population sizes were monitored using conidial numbers and colony forming units (CFU) per flower or fruit. A consistent population of P. frequentans, ranging from 10⁵ to 10⁶ number of conidia or 10³ to 10⁴ CFU of P. frequentans per flower or fruit, was obtained. Colonization of peach surfaces by all P. frequentans formulation are similar and it appears to follow a general pattern: (i) higher colonization of fruits at preharvest than on the flowers at bloom; (ii) high populations just after treatments, especially after preharvest treatments; and (iii) a slight decline between treatments, especially in cool and moist springs. The exponential model was the most appropriate for fitting and comparing the P. frequentans dynamic populations on peaches and nectarines over time. The linearization of the P. frequentans population curve was essential to determine dynamic population and for population levels forecast. A positive relationship between number of blossom and preharvest applications, temperature, relative humidity and dynamic of P. frequentans population applied on peaches was studied using a multiple regression model. Conclusions: Blossom and preharvest applications were the most important spray times for obtaining the highest population of P. frequentans on peach surfaces. Significance and Impact of the Study: The study provides useful information on dynamic P. frequentans population and its effects on the brown rot biocontrol.     

Population dynamics of Epicoccum nigrum, a biocontrol agent against brown rot in stone fruit

Aims:  To study the population dynamics of Epicoccum nigrum on peaches and nectarines and to enhance its colonization on fruit surfaces to improve its biocontrol efficacy against brown rot.
Methods and Results:  Twelve surveys were performed to study E. nigrum populations and their effect on the number of the pathogenic Monilinia spp. conidia in peach orchards in Spain and Italy between 2002 and 2005. Fresh conidia and five different formulations of E. nigrum conidia were applied three to six times to peach and nectarine trees from full flowering to harvest. The size of the E. nigrum populations was determined from the number of colony-forming units and conidial numbers per flower or fruit. Treatment with all conidial formulations increased the size of the indigenous conidial population on peach surfaces.
Conclusions:  Formulations of E. nigrum having high viability are most effective against conidia of the pathogen when applied at pit hardening and during the month immediately before fruit harvest.
Significance and Impact of the Study:  Application of an E. nigrum conidial formulation decreased the number of conidia of Monilinia spp. on fruit surfaces during the growing season to the same extent as fungicides.     

Relationship of in vitro and in planta screening: improving the selection process for biological control agents against Fusarium root rot in row crops

Fusarium root rot in row crops is typically managed by cultural practices and fungicide seed treatments. Biological control using microbial agents is another option but needs further development for improved disease management. Screening to identify biocontrol agents are crucial. However, relationships among the steps and how to improve the screening process are unresolved questions. Strains of Burkholderia (4), Bacillus (5) and Trichoderma (26) were studied in vitro against six Fusarium pathogens. All the bacteria and five selected Trichoderma strains were tested in planta in the greenhouse against diseases of Fusarium graminearum and Fusarium oxysporum. Burkholderia ambifaria C628, Bacillus simplex R180, and all Trichoderma isolates showed high reduction in disease levels in corn, soybean and wheat, ranging from 16 to 63%. Responses of the biocontrol agents during in vitro and in planta screening did not always correlate. In vitro and in planta tests should be considered independently in selecting biocontrol candidates.     

Solid state fermentation,storage and viability of Streptomyces similanensis 9X166 using agro-industrial substrates against Phytophthora palmivora-induced black rot disease in orchids

Streptomyces similanensis 9X166 is known to be an antagonist of the black rot pathogen of orchids, Phytophthora palmivora. In this study, we investigated the production of highly viable S. similanensis 9X166 cells by solid state fermentation using agro-industrial substrates, and the shelf life of a S. similanensis 9X166 dried solid. Rice bran was found to be the most appropriate raw material for production of both viable cells and β-1,3-glucanase. A medium containing 12?g of rice bran and coconut husks at a ratio of 10:2, supplemented with 10?mL of mineral salts produced the highest number of viable cells and greatest level of β-1,3-glucanase. Ammonium sulfate was the most suitable nitrogen source, and an initial moisture content of 65% and a temperature of 30°C were found to be optimal conditions for the production of viable cells and β-1,3-glucanase. Storing the dried fermented solid under non-vacuum conditions resulted in the highest cell viability. The specific rate of degradation on viability increased as the temperature increased to 37°C, according to the Arrhenius equation. There was no difference between the storage time estimated by the Arrhenius equation from the specific rate of degradation compared to the validated storage time of S. similanensis 9X166 dried solids when maintained at the ambient temperature in Thailand. At 60 days, the product retained 10⁶ CFU/g of S. similanensis 9X166 in dried solid, which was the minimal effective amount for 100% inhibition of P. palmivora in living orchids.     

A new oil-based formulation of Trichoderma asperellum for the biological control of cacao black pod disease caused by Phytophthora megakarya

In African cacao-producing countries, control of cacao black pod disease caused by Phytophthora megakarya is a priority. Introducing biological control agents as part of a P. megakarya control strategy is highly desirable, especially in a perspective of pesticide reduction. Trichoderma species are among the most used biological control agents. In Cameroon, Trichoderma asperellum formulated in wettable powder has produced positive effects against this disease. However, with this type of formulation, shelf-life and persistence of conidia on pods are limited. Our study therefore sought to develop a new T. asperellum formulation that would be more effective and better suited to the conditions of field application by small-scale producers in Cameroon. We selected a soybean oil-based oil dispersion, in which the half-life of the conidia reached 22.5 weeks, versus 5 weeks in aqueous suspension. Tested on detached pods, the formulation completely inhibited the development of the disease. When sprayed in the field on cacao clones highly sensitive to P. megakarya, the formulation resulted in 90% protection of treated pods after 1 week, and 50% after 3.2 weeks. The formulations exercised a measurable effect for up to 7 weeks, versus 2 weeks in the case of an aqueous conidial suspension and 5 weeks for that of a conventional fungicide (Kocide). Trichoderma asperellum formulated in oil dispersion has therefore great potential for the control of cacao black pod disease with less recourse to synthetic fungicides. Moreover, this formulation is well adapted to the types of sprayers used by small-scale cacao producers in Cameroon.     

Combination of antagonistic yeasts with two food additives for control of brown rot caused by Monilinia fructicola on sweet cherry fruit

AIMS: To evaluate beneficial effect of two food additives, ammonium molybdate (NH4-Mo) and sodium bicarbonate (NaBi), on antagonistic yeasts for control of brown rot caused by Monilinia fructicola in sweet cherry fruit under various storage conditions. The mechanisms of action by which food additives enhance the efficacy of antagonistic yeasts were also evaluated. METHODS AND RESULTS: Biocontrol activity of Pichia membranefaciens and Cryptococcus laurentii against brown rot in sweet cherry fruit was improved by addition of 5 mmol l(-1) NH4-Mo or 2% NaBi when stored in air at 20 and 0 degrees C, and in controlled atmosphere (CA) storage with 10% O2 + 10% CO2 at 0 degrees C. Population dynamics of P. membranefaciens in the wounds of fruit were inhibited by NH4-Mo at 20 degrees C after 1 day of incubation and growth of C. laurentii was inhibited by NH4-Mo at 0 degrees C in CA storage after 60 days. In contrast, NaBi did not significantly influence growth of the two yeasts in fruit wounds under various storage conditions except that the growth of P. membranefaciens was stimulated after storage for 45 days at 0 degrees C in CA storage. When used alone, the two additives showed effective control of brown rot in sweet cherry fruit and the efficacy was closely correlated with the concentrations used. The result of in vitro indicated that growth of M. fructicola was significantly inhibited by NH4-Mo and NaBi. CONCLUSION: Application of additives improved biocontrol of brown rot on sweet cherry fruit under various storage conditions. It is postulated that the enhancement of disease control is directly because of the inhibitory effects of additives on pathogen growth, and indirectly because of the relatively little influence of additives on the growth of antagonistic yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this study suggest that an integration of NH4-Mo or NaBi with biocontrol agents has great potential in commercial management of postharvest diseases of fruit.     

Efficacy of potent antagonistic yeast Wickerhamiella versatilis against soft rot disease of potato caused by Pectobacterium carotovorum subsp. carotovorum

Abstract

In this study, an antagonistic yeast isolate, Wickerhamiella versatilis was considered as a promising biocontrol agent against Pectobacterium carotovorum subsp. Carotovorum (Pcc) the causal agent of soft rot disease of potato. Antagonistic yeast inhibited the growth of Pcc in vitro, and reducing the soft rot severity of infected potato tubers (cv. Diamant) under greenhouse conditions. Consequently, cellulase and pectinase hydrolytic activities in infected potato tubers with yeast?+?Pcc were decreased compared with infected tubers with Pcc. The histological characterization of treated potato tubers with antagonistic yeast W. versatilis using scanning electron microscope showed the accumulation of extracellular substances that may induce plant resistant and protects potato tubers from hydrolysis and damages. This study may introduce the possibility of using the antagonistic yeast isolate, as a biocontrol agent against soft rot of potato tubers.     

Assessment of Boeremia exigua var. rhapontica,as a biological control agent of Russian knapweed (Rhaponticum repens)

Russian knapweed (Rhaponticum repens (L.) Hidalgo) is an herbaceous perennial weed that was introduced and has become invasive in the United States, particularly in the semi-arid west. It is characterized by its extensive root system, low seed production, and persistence. The weed has caused serious reductions in yields and crop value and may significantly devalue the land itself. Conventional control strategies have been inadequate because of the size of infestations and economic and environmental costs of control. Biological control has been a sought-after potential solution to this weed problem. In the summer of 2002, diseased R. repens plants were collected near Cankiri, Turkey, and the facultative saprophytic fungus Boeremia exigua isolate FDWSRU 02-059 was isolated from diseased plants. Bayesian analysis of the actin, beta-tubulin, calmodulin, elongation factor, and ITS genes, of 66 isolates, representing the ten species of Boeremia and the 11 varieties of B. exigua, including FDWSRU 02-059, showed that the isolate is a unique genetic entity and was named B. exigua var. rhapontica Berner, Woudenberg & Tunali, var. nov. MycoBank MB809363. Disease incidence and severity data from host-range determination tests conducted at 25 °C, the optimum temperature for growth and sporulation of B. ex. rhapontica, with adequate dew periods, were combined with a genetic distance matrix based on ITS sequences of 66 plant species related to R. repens. The combined disease and genetic data were analyzed by mixed model equations to produce best linear unbiased predictors (BLUPs), standard errors, and P > |t| values, in t-tests against zero, for disease incidence and severity for each species. BLUPs of disease incidence were significantly different from zero only for three Rhaponticum spp. while BLUPs of disease severity rankings were significantly different from zero only for R. repens, Rhaponticum carthamoides, Rhaponticum uniflorum, and Leuzea berardioides. Best linear unbiased predictors for differences in above-ground dry weights between control and inoculated plants of a subset of the species evaluated were not significant. However, above-ground damage by B. ex. rhapontica to R. repens was nearly twice that for any other species, except Rhaponticum species.     

Fungicidal control of damping-off and seedling root rot in Brassica species caused by Rhizoctonia solani in the growth chamber*

Eight fungicides (benodanil, carboxin, cyproconazole, fenpropimorph, fur-mecyclox, iprodione, pencycuron and tolclofos-methyl) were evaluated, under growth chamber conditions, as seed treatments against pre-emergence damping-off and post-emergence seedling root rot in six Brassica species. Five cultivars of B. rapa, four cultivars of B. juncea, four cultivars of B. napus and one cultivar/ strain from each of B. carinata, B. nigra and B. oleracea were grown in soilless mix infested with an isolate of Rhizoctonia solani AG-2-1. B. nigra and B. juncea were considerably less susceptible to R. solani than the four other species. Cyproconazole at 0.05-0.1 g a.i./kg seed and the other fungicides at 2–4 g a.i./ kg seed provided almost complete control of pre-emergence damping-off in most Brassica species and their cultivars. Their efficacy varied against the post-emergence seedling root rot. Furmecyclox, iprodione, tolclofos-methyl and pencycuron consistently gave good control of seedling root rot in all Brassica species and their cultivars. Benodanil and fenpropimorph provided moderate control, and carboxin and cyproconazole gave poor control against root rot. Efficacy of carboxin, cyproconazole, benodanil and fenopropimorph against seedling root rot varied significantly (P ≤ 0.05) among cultivars within a Brassica species.     

杨树球二孢溃疡病的研究—生物学特性、发病规律及药剂筛选

经试验,杨树球二孢溃疡病菌(Botryodiplodia populea Z.K.Zhong)适宜生长温度为20—25℃,pH6—7,在PDA和麦芽粉培养基生长良好。该病于4月中下旬产生病斑,5月上旬呈现颗粒状的子实体,5月中旬以后逐渐成熟开裂,溢出炭黑色的分生孢子,病原菌以菌丝体在树皮内越冬。经室内药剂筛选,660B生物农药抑菌效果最佳,可达95%。     

<Emphasis Type="Italic">Penicillium frequentans</Emphasis> isolated from<Emphasis Type="Italic"> Picea glehnii</Emphasis> seedling roots as a possible biological control agent against damping-off

Picea glehnii seedlings are affected by damping-off fungi in nurseries. The aims of this study were (1) to isolate fungi grown in the seedling rhizosphere in forest soil of P. glehnii, (2) to select fungi that produce antifungal compounds against Pythium vexans, and (3) to examine whether or not selected fungi can protect seedlings from P. vexans. Penicillium frequentans from Picea glehnii seedling roots produced antibiotic penicillic acid. Penicillic acid did not cause significant phytotoxicity to the seedlings. Penicillium frequentans increased the average percentage of surviving seedlings when inoculated together with Pythium vexans, but the increase was not significant. Vigorous mycelial growth of P. frequentans around seedling roots seems to be one of the mechanisms for protection, but the amount of penicillic acid was too low to show antifungal activity in the seedling rhizosphere.     

Predation of a biological control agent,Nesidiocoris tenuis (Hemiptera: Miridae), by the spider Leucauge blanda in greenhouse eggplant crops

Nesidiocoris tenuis (Hemiptera: Miridae) is used widely around the world as a biological control agent. In Kochi Prefecture, Japan, at the end of each greenhouse eggplant crop production period, the N. tenuis populations that have developed are collected and transferred to ‘natural-enemy-rearing greenhouses’ so that farmers can use the bug in the next production period. However, spider numbers have been increasing at the end of the production periods and it is becoming difficult to collect N. tenuis in some greenhouses. Therefore, we constructed specific primers for N. tenuis mtDNA to test whether the species was being preyed upon by the predominant spider species, Leucauge blanda. In polymerase chain reactions, these primers amplified a 148-bp fragment of the mitochondrial cytochrome oxidase subunit I gene of N. tenuis but not of any of the 13 other arthropod species tested that occurred in eggplant greenhouses. In a laboratory experiment, the rates of detection of N. tenuis DNA in the spiders after the end of a period of feeding on the bug were 90% at 0?h, 60% at 24?h, and 0% at 72?h. In a greenhouse field experiment, the rate of detection of N. tenuis DNA in the spider was 95%. These results suggest that L. blanda is responsible for the observed suppression of N. tenuis populations in greenhouse eggplant crops.     

Some dynamics of spread and infection by aeciospores of Puccinia punctiformis,a biological control pathogen of Cirsium arvense

Systemic disease of Cirsium arvense caused by Puccinia punctiformis depends on teliospores, from telia that are formed from uredinia, on C. arvense leaves. Uredinia result from infection of the leaves by aeciospores which are one main source of dispersal of the fungus. However, factors governing aeciospore spread, germination, infection, and conversion to uredinia and telia have not been extensively investigated. In this study, effective spread of aeciospores from a source area in a field was fitted to an exponential decline model with a predicted maximum distance of spread of 30 m from the source area to observed uredinia on one leaf of one C. arvense shoot. However, the greatest number of shoots bearing leaves with uredinia/telia was observed within 12 m of the source area, and there were no such shoots observed beyond 17 m from the source area. Aeciospore germination under laboratory conditions was low, with a maximum of about 10%. Temperatures between 18 °C and 25 °C were most favorable for germination with maximum germination at 22 °C. Temperature and dew point data collected from the Frederick, MD airport indicated that optimum temperatures for aeciospore germination occurred in late spring from about May 18 to June 20. Dew conditions during this period were favorable for aeciospore germination. A total of 122 lower leaves, 2 per shoot, on 61 C. arvense shoots were individually inoculated in a dew tent in a greenhouse by painting suspensions of aeciospores onto the leaves. Of these inoculated leaves, 47 produced uredinia within an average of 21.2 ± 6.9 days after inoculation. Uredinia were also produced, in the absence of dew, on 17 non-inoculated leaves of 12 shoots. These leaves were up to 4 leaves above leaves on the same shoots that had been individually and separately inoculated. Results of PCR tests for the presence of the fungus in non-inoculated leaves that were not bearing uredinia, showed that 44 leaves above inoculated leaves on 27 shoots were positive for the presence of the fungus. These leaves were up to 5 leaves above inoculated leaves on the same shoot. Uredinia production and positive PCR results on leaves above inoculated leaves on the same shoot indicated that aeciospore infection was weakly systemic. In other tests in which all leaves of plants were spray-inoculated with aeciospores, uredinia were produced by 10 days after inoculation and converted to telia and sole production of teliospores in about 63 days after inoculation. Successful systemic aeciospore infections in late spring would be expected to result in uredinia production in excess of a 1:1 ratio of aeciospore infections to uredinia and ultimately telia production in late summer. In this manner, systemic aeciospore infections would promote increased density of telia that lead to systemic infections of roots in the fall.     

Effect of certain cultural practices on susceptibility of potato tubers to soft rot disease caused by Erwinia carotovora pv. carotovora

Erwinia soft rot causes destructive and serious damage to many vegetable crops including potato in the field, transit and storage periods. The effect of certain cultural practices on the susceptibility of potato tubers to soft rot bacteria was studied and the results of this work can be summarised in the following: potato tubers harvested on 1 May first exhibited the highest disease incidence compared with those harvested on 15 May or 30 May. Harvesting on 15 June resulted in the lowest disease infection. The application of high levels of nitrogen fertiliser as urea (46.5%), ammonium nitrate (31%) and ammonium sulphate (20.5%) resulted in an increase of the susceptibility of potato tubers to bacterial soft rot disease. In contrast, the addition of phosphorous as superphosphate (15.5%) fertiliser caused the reverse effect. The addition of potassium as potassium sulphate (48%) alone at any of the tested levels showed no effect. The susceptibility of potato tubers to bacterial soft rotting disease was increased by increasing storage periods at 4°C for 1, 2, 3 and 4 months. Spraying copper sulphate exhibited the highest decrease in soft rot incidence disease followed by manganese, zinc and iron. However, spraying of boron increased susceptibility to the disease. Potato tubers obtained from plants sprayed with copper and stored for different periods showed the lowest susceptibility to disease incidence. Tuber sprayed with zinc, iron, manganese and finally boron came next.     

Genetic analysis of an introduced biological control agent reveals temporal and geographic change,with little evidence of a host mediated shift

The evolution of introduced biological control agents is largely un-explored. Although much is theorized, there is little empirical evidence quantifying the evolutionary dynamics of a biocontrol agent after release into a new environment. In this study we use Diachasmimorpha tryoni, a purposefully introduced biocontrol agent of Ceratitis capitata, to model and quantify spatial, temporal, and host-related evolutionary patterns. This parasitoid has undergone a host shift in its introduced environment, Hawaii, to the gall forming weed biocontrol agent, Eutreta xanthochaeta, an interaction likely mediated by competition for C. capitata with the egg-larval parasitoid Fopius arisanus. To elucidate potential evolutionary patterns we analyzed microsatellites and sequence data extracted from Hawaii and Australian population clusters defined by Structure, in Genepop, Canoco, and IBDWS. Our analysis revealed structuring of Hawaiian D. tryoni populations as defined by significant historic influences related to temporal structure, geographic space, host guild, and augmentative releases. The host-shift parasitoids were not genetically distinct from other Hawaii populations. There were small changes in microsatellite DNA at the population level, but only between Australia and Hawaii populations, not at the host level. These results show that D. tryoni has not undergone host-mediated evolution since introduction to Hawaii, despite the fact that they have expanded their host range in Hawaii to include the gall-forming E. xanthochaeta. To our knowledge this is the first study to quantify genetic differentiation of a biological control agent over geographic space and time using contemporary and museum specimens.     

Factors influencing colonization of cucumber roots by Clonostachys rosea f. catenulata,a biological disease control agent

The biocontrol fungus Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446, commercially available as Prestop® (Verdera Oy, Finland), is an effective antagonist against several root and foliar greenhouse pathogens. The biocontrol agent forms dense networks of hyphae on plant roots, grows internally in root epidermal cells, and produces hydrolytic enzymes, all of which lead to a reduction in pathogen propagules. An understanding of the environmental and host factors that influence root colonization by C. rosea f. catenulata is important to maximize disease control efficacy. Cucumber roots grown in nutrient solution in containers were inoculated with conidia of a GUS-transformed strain of C. rosea f. catenulata. Population levels associated with roots over time were assessed by colony-plate counts, GUS staining and enzymatic assays to determine GUS activity. Variables such as pH, temperature and growing medium were major factors that influenced population levels, while cucumber cultivar, addition of nutrients, and wounding of roots did not appear to significantly affect colonization. Population density of C. rosea f. catenulata on roots was highest when the nutrient solution was maintained at pH 5, 6, or 7, and at temperatures of 18–22°C. Lowest colonization levels were observed on roots of plants grown in potting mix or in field soil. Measurement of GUS activity provided a slightly more accurate assessment of root colonization levels compared to colony-plate counts. These results illustrate the optimal environmental conditions which can ensure maximum root colonization by C. rosea f. catenulata and enhance disease control by the biocontrol agent.     

杨树球二孢溃疡病的研究—生物学特性、发病规律及药剂筛选

经试验,杨树球二孢溃疡病菌(Botryodiplodia populea Z.K.Zhong)适宜生长温度为20—25℃,pH6—7,在PDA和麦芽粉培养基生长良好。该病于4月中下旬产生病斑,5月上旬呈现颗粒状的子实体,5月中旬以后逐渐成熟开裂,溢出炭黑色的分生孢子,病原菌以菌丝体在树皮内越冬。经室内药剂筛选,660B生物农药抑菌效果最佳,可达95％。