Altered nitrogen metabolism in biocontrol strains of Penicillium rubens.

The importance of the metabolic route of nitrogen in the fungus Penicillium rubens (strain PO212) is studied in relation to its biocontrol activity (BA). PO212 can resist a high concentration of chlorate anion and displays a classical nitrate-deficiency (nit-) phenotype resulting in poor colonial growth when nitrate is used as the main source of nitrogen. Analyses of genes implicated in nitrate assimilation evidenced the strong sequence conservation of PO212 and CH8 genome with penicillin producers such as reference strain P. rubens Wisconsin 54-1255, P2niaD18 and Pc3, however also revealed the presence of mutations. PO212 carries a mutation in the gene coding for zinc-binuclear cluster transcription factor NirA that specifically mediates the regulation of genes involved in nitrate assimilation. The nirA1 mutation causes an early stop of NirA factor, losing 66% of its sequence. The NirA1 mutant form is unable to mediate a nitrate-dependent regulation of nitrate and nitrite reductase coding genes. In this study, we study another isolate, CH8, with potential BA and nit- phenotype. A mutation in the nitrate permease coding gene crnA was found in CH8. An insertion of a guanine in the coding sequence cause a frameshift in CrnA with the loss of the last two transmembrane domains. Analysis of PO212 and CH8 isolates and complementation strains show the importance of NirA regulator in maintaining correct transcriptional levels of nitrate and nitrite reductases and suggest CrnA as the main nitrate transporter. the presence of alternative transporter for chlorate and the existence of a mechanism for preventing nitrite derived toxicity in Penicillum. BA of PO212 is partially altered when nirA1 mutation was complemented. This result and the finding of CH8, a novel biocontrol P. rubens strain with a nit- phenotype, suggest that nitrogen metabolism is a component of biocontrol capacity.

Characterization of Fusarium solani Populations Associated with Spanish Strawberry Crops.

Fusarium solani is an emerging pathogen reported on Spanish strawberry crops both in nurseries and in fruit production fields, causing wilt and root rot. Pathogenicity, morphocultural characteristics, and sensitivity to biocides of 103 F. solani isolates recovered from symptomatic strawberry plants and soils from both Spanish strawberry areas were determined. The differences of isolates within and between nurseries and field crops in relation to these parameters were analyzed. Considerable variability in morphological and pathogenic characteristics was observed among the isolates in both areas. The majority of isolates were not pathogenic (62%), and only 38 F. solani isolates (37.62%) caused disease on strawberry plants under controlled conditions; 52.63% of pathogenic isolates induced low severity symptoms. Almost 70% of pathogenic isolates caused stunting on plants. The morphological characters that best explain the F. solani variability (86.85%) were colony color and the presence of macroconidia on culture medium. The sensitivity to the fumigants tested was similar between the isolates from nurseries and fruit production fields, showing greater sensitivity to the field doses of dazomet and chloropicrin. However, the isolates were less sensitive to metam sodium and poorly sensitive to 1,3-dichloropropene. This work can contribute to the advancement of sustainable production of strawberry.

Detection of Latent Monilinia Infections in Nectarine Flowers and Fruit by qPCR.

Most stone fruit with a latent brown rot infection caused by Monilinia do not develop visible signs of disease until the arrival of fruit at the markets or the consumer's homes. The overnight freezing-incubation technique (ONFIT) is a well-established method for detecting latent brown rot infections, but it takes between 7 to 9 days. In this report, we inform on the advantages of applying a qPCR-based method to (i) detect a latent brown rot infection in the blossoms and fruit of nectarine trees (Prunus persica var. nucipersica) and (ii) distinguish between the Monilinia spp. in them. For applying this qPCR-based method, artificial latent infections were established in nectarine flowers and fruit using 10 Monilinia fructicola isolates, 8 M. fructigena isolates, and 10 M. laxa isolates. We detected greater amounts of M. fructicola DNA than M. laxa and M. fructigena DNA in latently infected flowers using qPCR. However, greater DNA amounts of M. laxa than M. fructicola were detected in the mesocarp of latently infected nectarines. We found that the qPCR-based method is more sensitive, reliable, and quicker than ONFIT for detecting a latent brown rot infection, and could be very useful in those countries where Monilinia spp. are classified as quarantine pathogens.

Fruit maturity and post-harvest environmental conditions influence the pre-penetration stages of Monilinia infections in peaches.

Brown rot caused by the fungi Monilinia laxa (Aderhold and Ruhland) Honey, M. fructicola (Winter) Honey, or M. fructigena (Aderhold and Ruhland) is a serious fungal disease of peaches. The fungal infection process begins when fungal conidia germinate on the fruit surface to produce germ tubes and/or appressoria, and the incidence of brown rot increases as fruit approaches maturity. The interaction between the fungal infection process, peach maturity, and the environmental conditions is not well understood. Accordingly, the objectives of this investigation were to investigate germ tube and appressorial formation by M. laxa and M. fructicola when they were exposed to peach skin from mature and immature fruit at various temperatures and relative humidities (RHs). The greatest number of germ tubes was found when M. laxa or M. fructicola was incubated in culture medium which contained a skin extract of mature peaches. In contrast, the greatest number of appressoria was found when M. laxa or M. fructicola was incubated in culture medium which contained a skin extract of immature peaches. Although M. fructicola produced the same number of germ tubes and appressoria at 4°C, M. fructicola produced more germ tubes than appressoria at temperatures higher than 10°C. M. laxa produced more germ tubes than appressoria at any temperature, except when it was incubated for 48h on culture medium which contained a skin extract of immature peaches at 10°C at 80% or 100% RH, or at 25°C at 60% RH. M. laxa conidia germinated better than M. fructicola conidia at low temperatures. Germ tube and appressorial formation by Monilinia spp. were influenced by fruit postharvest handling. The number of germ tubes that were formed by M. laxa conidia was significantly greater than that for M. fructicola when the conidia were incubated at 100% RH, and this number increased after 3days of refrigeration. The number of appressoria that were formed by both Monilinia spp. also increased after 3 consecutive days of refrigeration. Negligible or no germination of M. fructicola and M. laxa conidia occurred when the RH was 60%. We concluded that the dissimilar abilities of M. laxa and M. fructicola to germinate and form appressoria at low temperatures conferred a competitive advantage to M. laxa to survive during fruit postharvest refrigeration and cold storage at 4°C.

Growth and aggressiveness factors affecting Monilinia spp. survival peaches.

Brown rot of stone fruit is caused by three species of Monilinia, Monilinia laxa, M. fructigena, and M. fructicola. Eleven components of 20 different isolates of each of the three Monilinia species were analyzed to determine distinct aggressiveness and growth characteristics among the three fungi. M. fructicola showed the greatest lesion diameter, and the lowest incubation and latency period on fruit postharvest, however isolates of M. fructigena exhibited less aggressiveness components. Five growth characteristics of M. fructicola could be used to distinguish M. fructicola from the other two species. The dendrogram generated from only the presence of sclerotia and lesion length on infected fruit separated the 60 isolates into two clusters (r=0.93). One cluster was composed of the M. laxa and M. fructigena isolates and the other cluster comprised the M. fructicola isolates. However, the dendrogram generated based on the presence of stromata and sclerotia in the same colony of the three species when they were grown on potato dextrose agar, and the lesion diameter on fruit infected with each species separated the 60 isolates into three clusters (r=0.81). Each cluster comprised the isolates of each of three Monilinia spp. We discussed the effect of M. fructicola growth and aggressiveness differences on the displacement of M. laxa and M. fructigena by M. fructicola recorded in Spanish peach orchards and their effect on brown rot at postharvest.

Growth and aggressiveness factors affecting Monilinia spp. survival peaches.

Brown rot of stone fruit is caused by three species of Monilinia, Monilinia laxa, M. fructigena, and M. fructicola. Eleven components of 20 different isolates of each of the three Monilinia species were analysed to determine distinct aggressiveness and growth characteristics among the three fungi. M. fructicola showed the greatest lesion diameter, and the lowest incubation and latency period on fruit postharvest, however isolates of M. fructigena exhibited less aggressiveness components. Five growth characteristics of M. fructicola could be used to distinguish M. fructicola from the other two species. The dendrogram generated from only the presence of sclerotia and lesion length on infected fruit separated the 60 isolates into two clusters (r=0.93). One cluster was composed of the M. laxa and M. fructigena isolates and the other cluster comprised the M. fructicola isolates. However, the dendrogram generated based on the presence of stromata and sclerotia in the same colony of the three species when they were grown on potato dextrose agar, and the lesion diameter on fruit infected with each species separated the 60 isolates into three clusters (r=0.81). Each cluster comprised the isolates of each of three Monilinia spp. We discussed the effect of M. fructicola growth and aggressiveness differences on the displacement of M. laxa and M. fructigena by M. fructicola recorded in Spanish peach orchards and their effect on brown rot at postharvest.

Penicillium oxalicum reduces the number of cysts and juveniles of potato cyst nematodes.

AIM: To test the biocontrol potential of Penicillium oxalicum, a biocontrol agent against fungal diseases and against the potato cyst nematodes (PCNs), Globodera pallida and Globodera rostochiensis. METHODS AND RESULTS: We tested the effect of P. oxalicum on the nematode cysts under laboratory conditions or in soil microcosms. A reduction in the rate of G. pallida juveniles hatching by P. oxalicum was observed when root diffusates from the 'Monalisa' and the 'Désirée' potato cultivar were used (98·6 and 74·1% reduction, respectively). However, the rate of G. pallida juveniles hatching was not significantly reduced when root diffusates from the 'San Pedro' tomato cultivar were used. Penicillium oxalicum also significantly reduced the ability of the G. rostochiensis juveniles to hatch (30·9% reduction) when root diffusates of the 'Désirée' potato cultivars were used. Penicillium oxalicum treatment of the soil significantly reduced the number of G. pallida cysts that were recovered from the soil of each pot that contained the 'Désirée' potato cultivar. CONCLUSIONS: Our results show that P. oxalicum is a potential biocontrol inoculant for protecting potato crops against PCNs. SIGNIFICANCE AND IMPACT OF THE STUDY: Penicillium oxalicum has potential to be used in order to reduce PCNs.

Potential of Spanish sour-sweet pomegranates (cultivar C25) for the juice industry.

Commercial juices of sweet pomegranates and fresh juices of sour-sweet pomegranates were analysed for organic acids, sugars, antioxidant activity, volatile composition, sensory profile and consumer liking. Organic acids and sugars were analysed by high-performance liquid chromatography, while volatiles were extracted using hydrodistillation and analysed by gas chromatography-mass spectrometry and gas chromatography-flame ionisation detector. Malic acid was predominant in sweet juices while citric acid in sour-sweet samples. Fructose and glucose were found as the predominant sugars in all juices. A total of 18 compounds were found in pomegranate aroma profile, including monoterpenes, aldehydes, alcohols, monoterpenoids and linear hydrocarbons; the most abundant compounds were 3-carene, α-terpinene and α-terpineol. The total concentration of volatiles ranged from 2.0 up to 5.1 mg/L, with commercial samples presenting higher total concentrations due to the presence of peel volatiles. The high potential of sour-sweet pomegranate fruits for the juice industry was supported by (a) the high values of positive attributes, such as colour and fresh pomegranate flavour and (b) the high overall liking of consumers.

Enhancing the adhesion of Epicoccum nigrum conidia to peach surfaces and its relationship to the biocontrol of brown rot caused by Monilinia laxa.

AIMS: To find a formulation of Epicoccum nigrum conidia that enhances its adhesion to peach surfaces and improves its biocontrol efficacy against brown rot caused by Monilinia laxa. METHODS AND RESULTS: The stickers, glycerol, sodium alginate and methylcellulose; the desiccants, silica powder and talc; and a commercial adhesive (NU FILM 17) were added at two different points during the production of an E. nigrum conidial formulation to improve conidial adhesion to peach surfaces. Conidial adhesion levels were determined from the number of E. nigrum conidia that adhered to glass slides or peach surfaces and conidial viability of adherent E. nigrum conidia was determined from the number of colony-forming units of glass or peach-adherent E. nigrum that grew on Petri dishes that contained potato dextrose agar. Compared to dried E. nigrum conidia without additives, the adhesion and viability of adherent E. nigrum conidia to peach surfaces were enhanced when either 1.25% sodium alginate or 2.5% methylcellulose was added to the conidial mass after fluid-bed drying, and when 2.5% methylcellulose was added to the conidial mass after its production and before fluid-bed drying. Epicoccum nigrum conidial formulations with 2.5% methylcellulose were more effective than dried E. nigrum conidia without additives in reducing the incidence of brown rot in peaches caused by M. laxa. CONCLUSIONS: When 2.5% methylcellulose is incorporated into an E. nigrum conidial formulation, the adhesion of E. nigrum conidia to peach surfaces improves and results in efficacious biocontrol of brown rot. SIGNIFICANCE AND IMPACT OF THE STUDY: A new improved formulation of a biocontrol agent has been developed to improve the control of M. laxa on peaches.

Primary Inoculum Sources of Monilinia spp. in Spanish Peach Orchards and Their Relative Importance in Brown Rot.

Immediately following the identification of Monilinia fructicola in a Spanish peach orchard in the Ebro Valley in 2006, this orchard and two other orchards in the same valley were intensively sampled for potential tree and ground sources of primary Monilinia inoculum before and during three growing seasons between 2006 and 2008. Overwintered Monilinia spp. produced inoculum from only mycelium, and no apothecia were found in any of the three orchards over the three growing seasons. Mummies on trees were the main source of primary inoculum. More than 90% of Monilinia isolates on all fruit mummies were M. laxa. Positive relationships were found between (i) the number of mummified fruit and the incidence of postharvest brown rot (P = 0.05, r = 0.75, n = 8), and (ii) the number of mummified fruit and nonabscised aborted fruit in the trees and the number of conidia on the fruit surface (P = 0.04, r = 0.71; P = 0.01, r = 0.94, respectively, n = 8) and the incidence of latent infection (P = 0.03, r = 0.75; P = 0.001, r = 0.99; respectively, n = 8). In addition, the numbers of mummified fruit and pruned branches on the orchard floor were correlated with the number of airborne conidia in the orchard. Based on the results of these surveys, the control of brown rot in stone fruit orchards is discussed.

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.

Development of a Method for Detection of the Biocontrol Agent Penicillium oxalicum Strain 212 by Combining PCR and a Selective Medium.

The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Penicillium oxalicum strain 212 (PO212) is being developed for the control of tomato pathogens. In this study, we demonstrated that PO212 was more effective for controlling Fusarium oxysporum f. sp. lycopersici in tomato plants than 13 other P. oxalicum strains. A new semiselective medium was developed as a preliminary screen for P. oxalicum from soil. This semiselective medium was a modified Fusarium selective medium that contained 0.006 g of nystatin per liter. The growth of P. oxalicum strain 212 was not inhibited on this medium, but it did inhibit the growth of 11 fungal species. Specific identification of the biocontrol strain and its quantification were achieved using a polymerase chain reaction with a strain-specific pair of primers (POITS1F/POITS2R1) and dilution plating. This primer set differentiated the biocontrol strain from 13 other strains of P. oxalicum. There were differences in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA of 25 strains of P. oxalicum and those of PO212. Based on the differences in the nucleotide sequences of the ITS regions in rDNA of PO212 and other P. oxalicum strains, a relationship between the nucleotide sequences in the ITS region and biocontrol efficacy is postulated.

First Report of Brown Rot Caused by Monilinia fructicola in Peach Orchards in Ebro Valley, Spain.

Monilinia fructicola causes brown rot of stone fruit in India, Japan, the Republic of Korea, Oceania, and North and South America and is in the A2 list of quarantine organisms for Europe. M. fructicola was found in peach orchards for the first time in Europe in 2001 in France (4) and later in the Czech Republic (2). M. fructicola was not detected among 428 isolates of Monilinia spp. collected from Spanish peach orchards from 1998 to 2005. In March of 2006, M. fructicola was detected to be overwintering in three mummified peach fruit (cv. Autumn Free) trees in an orchard located in Sudanell (Lleida, Spain). Morphological and molecular identification of isolates were performed according to protocols previously described (1,3). The characteristics of these isolates were: i) colonies were entire and showing concentric rings of spores when grown on potato dextrose agar (PDA); ii) sporogenous tissues were gray to buff; iii) single and nearly straight germ tubes were at least 220 µm long before branching; and iv) growth rates on PDA under long-wave UV/darkness were as much as 20 × 10 mm2. Isolates were further identified by a PCR test using primers developed with sequence-characterized amplification region markers obtained by random amplified polymorphic DNA for M. fructicola: IColaS (GAGACGCACACAGAGTCAG) and IColaAS (GAGACGCACATAGCATTGG) (3). The expected PCR product of 386 bp was produced only in M. fructicola isolates. Koch's postulates were fulfilled with the three isolates by inoculating five healthy fruit with a conidial suspension of each isolate (104 conidia ml-1). Symptoms similar to those observed in the field were small brown spots, which rapidly showed brown rot. Noninoculated control fruit did not show symptoms. The fungus was reisolated on PDA from inoculated fruit after 4 days of incubation at 22°C, 80 to 100% relative humidity, and 16 h under fluorescent lighting, 100 µE·m-2·s-1. To our knowledge, this is the first report of M. fructicola in peach orchards in Spain. References: (1) A. De Cal and P. Melgarejo. Plant Dis. 83:62, 1999. (2) J. Duchoslavová et al. Plant Dis. 91:907, 2007. (3) I. Gell et al. J. Appl. Microbiol. 103:2629, 2007. (4) J. Lichou et al. Phytoma 547:22, 2002.

Influence of additives on adhesion of Penicillium frequentans conidia to peach fruit surfaces and relationship to the biocontrol of brown rot caused by Monilinia laxa.

Additives, such as sucrose, d-sorbitol, glycerol, sodium alginate, carboxymethyl cellulose, silica gel, gelatine, non-fat skimmed milk and a commercial adhesive were added to conidia of Penicillium frequentans at two different points in the production process of the formulation of this fungus to improve conidial adhesion. Conidial adhesion was estimated as the number of P. frequentans conidia (no. conidia cm(-2)) and colony-forming units of P. frequentans per unit area (cfu cm(-2)) that adhered to glass slides or to peach surfaces. The P. frequentans conidial concentration had a significant effect on conidial adhesion, while the shelf life of conidia did not have any effect. The highest adhesion of P. frequentans conidia to glass slides was observed when conidial concentrations were greater than 10(6) conidia ml(-1). P. frequentans conidial adhesion was improved when 1.5% sodium alginate or 1.5% carboxymethyl cellulose were added to the conidial mass obtained after production and before drying by the fluid bed drying process. Conidial adhesion was also enhanced when 1.5% sodium alginate, 1.5% carboxymethyl, or 1.5% gelatine were added to conidia after fluid bed drying. P. frequentans formulations with 1.5% sodium alginate or 1.5% carboxymethyl cellulose were more effective in reducing brown rot caused by Monilinia laxa than dried P. frequentans conidia alone. Our results show that additives can improve adhesion of P. frequentans conidia to fruit surfaces, resulting in more effective control of brown rot in peaches.

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 10(5) to 10(6) number of conidia or 10(3) to 10(4) 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.

Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit trees.

AIMS: To design a protocol for the universal diagnosis of brown rot by polymerase chain reaction (PCR) in plant material and subsequently Monilinia spp. identification. METHODS AND RESULTS: Primers for discrimination of Monilinia spp. from other fungal genera by PCR were designed following a ribosomal DNA analysis. Discrimination among species of Monilinia was subsequently achieved by developing primers using SCAR (Sequence Characterised Amplified Region) markers obtained after a random amplified polymorphic DNA study. In addition, an internal control (IC) based on the utilization of a mimic plasmid was designed to be used in the diagnostic protocol of brown rot to recognize false negatives due to the inhibition of PCR. CONCLUSIONS: The four sets of primers designed allowed detection and discrimination of all Monilinia spp. causing brown rot in fruit trees. Addition of an IC in each PCR reaction performed increased the reliability of the diagnostic protocol. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection protocol presented here, that combined a set of universal primers and the inclusion of the plasmid pGMON as an IC for diagnosis of all Monilinia spp., and three sets of primers to discriminate the most important species of Monilinia, could be an useful and valuable tool for epidemiological studies. The method developed could be used in programmes to avoid the spread and introduction of this serious disease in new areas.

Effects of stabilizers on shelf-life of Epicoccum nigrum formulations and their relationship with biocontrol of postharvest brown rot by Monilinia of peaches.

AIM: To find a formulation of Epicoccum nigrum conidia that maintains a high viability over time and which proves efficient to biocontrol peach rot caused by Monilinia spp. METHODS AND RESULTS: We tested the effect of stabilizers and desiccants on the shelf-life of Epicoccum nigrum conidia. Conidial samples were dried for 40 min at 40 degrees C in a fluidized bed-dryer to obtain moisture contents <15%. The toxicity of additives was tested by assaying production of conidia in fermentations and germinability of the produced conidia: 50% PEG300, 10%-5% KCl (stabilizers) and 95.24% Cl(2)Ca (desiccant) significantly (P = 0.05) reduced conidial germination. To enhance shelf-life of dried conidia, nontoxic stabilizers were added at the following different stages of the production-drying process: (i) to substrate contained in bags before production, (ii) to conidial centrifuge pellets obtained after production, before filtering and drying, (iii) to conidial centrifuge pellets obtained after production, before adding talc and drying, and (iv) to conidial centrifuge pellets obtained after production, before adding silica powder and drying. Conidial germinability was tested at 0, 180 and 365 days after storage at room temperature. Shelf-life of formulations retaining the highest viability were conidia produced with 1% KCl or 50% PEG 8000, conidia dried with 2.5% methylcellulose, and conidia dried with 1% KCl + silica powder. All these formulations improved the shelf-life of E. nigrum conidia and significantly reduced brown rot on peaches. CONCLUSIONS: Our results show that additives improve the shelf-life of E. nigrum and assist controlling brown rot on peaches. SIGNIFICANCE AND IMPACT OF THE STUDY: New improved formulations of a biocontrol agent have been obtained which will improve the control of Monilinia on peach.

Effect of stabilizers on the shelf-life of Penicillium frequentans conidia and their efficacy as a biological agent against peach brown rot.

Stabilizers were added to conidia of Penicillium frequentans at two different points of the production-formulation process to improve shelf-life of conidia stored at different temperatures. Effects were also tested on conidial germination and production. Germination of conidia without additives was 90.2%; sodium chloride, potassium chloride, triton TX100, dimethyl sulphoxide, peroxidase, 0.375% and 0.075% ascorbic acid, 7.5% and 3.75% sucrose, and 7.5% and 3.75% d-sorbitol reduced significantly (P=0.05) conidial germination, while no effect was observed with glucose, lactose, maltose, sodium glutamate, glycerol, peptone, sodium alginate, carboximethylcellulose, Tween 80, and gelatine. Production of P. frequentans conidia in solid-state fermentation without additives was 1.07 conidia x 10(8) g(-1) of dry substrate. The highest tested doses of glucose, lactose, maltose, sodium glutamate, and glycerol enhanced production of P. frequentans, while the lowest tested doses of d-sorbitol and ascorbic acid reduced it. No significant effect was observed with sucrose, peptone, sodium alginate, carboximethylcellulose, gelatine and Tween 80. Conidial germinability after one year of storage at different temperatures was studied in some formulations. It was lower than 18% after 365 days of storage at room temperature in control samples (without any additive), being enhanced when 7.5% glucose, 7.5% glycerol, or 1.5% sodium alginate was added to the substrate in bags before fermentation; or when 7.5% glucose, 7.5% sodium glutamate, or 1.5% sodium alginate was added to conidia before drying. Germinability of conidia produced without any additive and stored at 4 degrees C was significantly higher (38%) than at room temperature, being enhanced when 7.5% glycerol or 1.5% sodium alginate was added to the substrate in bags before fermentation; and when 7.5% glucose, or 1.5% sodium alginate was added to conidia before drying. No effect was observed with the presence or absence of light or high vacuum. Four formulations of P. frequentans conidia reduced disease incidence by more than 55%. The relationship of the disease control with the viability of P. frequentans was discussed.

Dispersal Improvement of a Powder Formulation of Penicillium oxalicum, a Biocontrol Agent of Tomato Wilt.

Sugars, polyalcohols, inorganic salts, and detergents were added to conidia of Penicillium oxalicum at three different points of the production-formulation process to improve water dispersal. Effects also were tested on conidial germination and production. Conidial germination without additives ranged from 51 to 79%. Additives did not reduce conidial germination except for 50% polyethylene glycol (PEG) 300 and 10% CaCl2. Sunflower oil and sodium alginate, sucrose (0.5, 15, 30, and 60%), D-sorbitol (30 and 60%), glycerol (2, 5, 20, and 30%), 30% PEG 300, CaCl2 (0.01 to 1%), Tween 20 (0.01, 0.02, 0.5, and 1%), and Tween 80 (0.01 to 1%) enhanced conidial germination. Production without additives ranged from 0.57 to 4.58 conidia × 108 g-1 substrate. Additives did not affect conidial production except for reduction by 60% D-sorbitol, 60% fructose, and 10% CaCl2. Conidial dispersal in water improved when 1.5% sodium alginate was added to substrate in bags before production, and when 1.5% sodium alginate, 60% sucrose, 60% D-sorbitol, 60% fructose, 5 to 20% PEG 8000, or 20% glycerol were added to conidia before drying. Dispersal of dried conidia was enhanced with 1% Tween 20, 1% Tween 80, 1% Trition X-100, 10% Agral, and 1.5% sunflower oil. Two P. oxalicum formulations (conidial suspensions maintained with 60% sucrose or 1.5% sodium alginate for 10 min before drying) significantly reduced tomato wilt caused by Fusarium spp. under greenhouse conditions and, in a preliminary trial, by Verticillium spp. in a field assay.

Solid substrate production of Epicoccum nigrum conidia for biological control of brown rot on stone fruits.

Production of conidia of Epicoccum nigrum, a biocontrol agent of the fungal pathogen Monilinia laxa, was tested in liquid- and solid-state fermentation. Liquid fermentation was conducted in 250 ml Erlenmeyer flasks containing 50 ml of a mineral medium (containing per litre: 20 g lactose, 10 g NO3K, 1 g K2HPO4, 0.5 g MgSO4.7H2O, and 1 ml of a minor-element solution), inoculated with 2 x 10(5) E. nigrum conidia ml(-1), and incubated at 20-25 degrees C and 150 rpm for 7 days. Solid-state fermentation was carried out in specially designed plastic bags (600 cm3) (VALMIC) containing either 50 g of peat/vermiculite (1:1, w/w), or 50 g of peat/vermiculite/lentil meal (1:1:1, w/w/w) with 40% (v/w) initial moisture content. Substrate was inoculated with a conidial suspension of E. nigrum to give 10(5) conidia g(-1) substrate, and bags were incubated at 20-25 degrees C for 7 days in darkness. The amount of conidia of E. nigrum obtained in solid-state fermentation with substrate based on peat/vermiculite/lentil meal was 10-fold higher than with substrate based on peat/vermiculite or in liquid fermentation. Conidial production under these conditions was maintained in the range of 10(8) conidia g(-1) substrate from 10 to 150 days after inoculation. Germinability of these conidia was >90%. Addition of other nutrients than lentil meal to peat/vermiculite did not enhance production of conidia. Presence of peat in the substrate was necessary for good conidia production, but change in the kind of peat or vermiculite did not improve conidial production. Conidial production was similar when the substrate was inoculated with 10(5), 10(6) or 10(7) conidia g(-1) dry substrate. Incubation of bags in light conditions did not enhance conidial production. Fresh conidia produced in this solid-state fermentation system reduced the incidence and lesion diameter induced by M. laxa on peaches.