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

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

Analysis of genetic diversity in <Emphasis Type="Italic">Monilinia fructicola</Emphasis> from the Ebro Valley in Spain using ISSR and RAPD markers

The genetic diversity of Spanish and French field populations of Monilinia fructicola, a quarantine fungal pathogen in Europe, was compared with that of Californian, Uruguayan, and New Zealand M. fructicola populations using inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers. Unweighted pair-group method with arithmetic average (UPGMA) cluster analysis and principal component analysis (PCA) of the ISSR data set revealed that the Spanish and French M. fructicola isolates were more closely related between themselves than to the non-European isolates. The levels of genetic diversity in the Spanish and French isolates are lower than those of the non-European isolates, indicating that M. fructicola is a recently introduced pathogen. UPGMA cluster analysis and PCA of the combined ISSR + RAPD data set of the European M. fructicola populations revealed that the Spanish isolates were more closely related among themselves than with the French isolates. Analysis of molecular variance partitioned the genetic variance to among the two regions (Spain and France) (20%), among the regional populations (35%), and within the populations in each region (45%) suggesting restricted gene flow between the three European populations. The observed index of association (I_A) in each European M. fructicola populations indicates that the French and Spanish populations of M. fructicola are mainly asexually reproducing, with the Sudanell population potentially having a teleomorphic stage. The present finding of low genetic diversity in the Spanish and French M. fructicola populations is probably due to founder effects and genetic drift.     

Development of a multiplex PCR for the identification of Fusarium solani and F. oxysporum in a single step

Journal of Plant Diseases and Protection - Fusarium solani and F. oxysporum are plant pathogenic fungi that cause root rot and wilt, respectively, in many economically important crops....     

A rapid diagnostic assay for detection and quantification of the causal agent of strawberry wilt from field samples

Verticillium dahliae Kleb, the cause of Verticillium wilt disease, is a destructive pathogen that leads to severe yield losses in strawberry fields and thus considerable economic damages. Although rapid identification and detection methods are becoming available more, pathogen quantification remains one of the main challenges in the disease management. In this study, a real-time polymerase chain reaction (rtPCR) assay was developed to quantitatively assess V. dahliae abundance directly from affected roots and soil collected from different areas in Estonia. A specific primer pair based on the ribosomal DNA (rDNA) internally transcribed spacer was designed for SYBR Green-based assay. Strawberry plant and soil samples were randomly collected from different areas in Estonia and analyzed for V. dahliae by soil plating technique and rtPCR assay. The assay was specific for V. dahliae so that the minimum detection limit was 0.93?pg?µl^?1 of pathogen DNA and the lowest amount of V. dahliae detected in soil was 10.48?pg?µl^?1 of target DNA corresponding to one microsclerotia per gram of soil. This technique allowed rapid detection and quantification of the pathogen DNA at the picogram level in soils and even in symptomless plants, facilitating the screening of the pathogen in diverse areas. This is the first study about the rtPCR technique being used successfully to assess populations of V. dahliae with high specificity and sensitivity in Estonia strawberry fields. Results of this research can be useful for growers and agricultural organizations to improve available disease management strategies against Verticillium wilt.