Abstract: ABSTRACT A sensitive nested-polymerase chain reaction (PCR) protocol was developed using either of two primer pairs that improves the in planta detection of Peronospora arborescens DNA. The new protocol represented an increase in sensitivity of 100- to 1,000-fold of detection of the oomycete in opium poppy tissue compared with the detection limit of single PCR using the same primer pairs. The new protocol allowed amplification of 5 to 0.5 fg of Peronospora arborescens DNA mixed with Papaver somniferum DNA. The protocol proved useful for amplifying Peronospora arborescens DNA from 96-year-old herbarium specimens of Papaver spp. and to demonstrate that asymptomatic, systemic infections by Peronospora arborescens can occur in wild Papaver spp. as well as in cultivated opium poppy. Also, the increase in sensitivity of the protocol made possible the detection of seedborne Peronospora arborescens in commercial opium poppy seed stocks in Spain with a high frequency, which poses a threat for pathogen spread. Direct sequencing of purified amplicons allowed alignment of a Peronospora arborescens internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequence up to 730-bp long when combining the sequences obtained with the two primer sets. Maximum parsimony analysis of amplified Peronospora arborescens ITS rDNA sequences from specimens of Papaver dubium, P. hybridum, P. rhoeas, and P. somniferum from different countries indicated for the first time that a degree of host specificity may exist within populations of Peronospora arborescens. The reported protocol will be useful for epidemiological and biogeographical studies of downy mildew diseases as well as to unravel misclassification of Peronospora arborescens and Peronospora cristata, the reported causal agents of the opium poppy downy mildew disease.

Abstract: ABSTRACT Fusarium oxysporum f. sp. ciceris, and the root-knot nematode Meloidogyne artiellia, coinfect chickpea crops in several countries of the Mediterranean Basin. The influence of root infection by M. artiellia on the reactions of chickpea genotypes with different reaction to infection with F. oxysporum f. sp. ciceris races 0, 1A, and 2 was investigated under controlled environmental conditions. Results demonstrated that co-infection of chickpea genotypes resistant to specific fungal races by M. artiellia did not influence the Fusarium wilt reaction of the plant, irrespective of the F. oxysporum f. sp. ciceris race assayed. However, in some of the assayed combinations, coinfection by both pathogens significantly affected the level of colonization by the fungus or reproduction of the nematode in the root system. Thus, coinfection of chickpea plants with Foc-0 and M. artiellia significantly decreased the level of colonization of the root system by F. oxysporum f. sp. ciceris in genotypes 'CA 336.14.3.0' and 'PV 61', but not in 'ICC 14216 K' and 'UC 27'. Similarly, the nematode reproduction index was also significantly reduced by coinfection with Foc-0 in the four chickpea genotypes tested and inoculated with this race. Conversely, coinfection of chickpea plants with Foc-1A and M. artiellia significantly increased colonization of the root system by the fungus in all genotypes inoculated with this race, except for line BG 212. Altogether, we confirmed the complete resistance phenotype of 'UC 27' and 'ICC 14216 K' to Foc-0, and of 'ICC 14216 K' to Foc-1A and Foc-2, and demonstrated that this resistance was not modified by coinfection of the resistant plant with M. artiellia.

Abstract: ABSTRACT The development of Verticillium wilt epidemics in olive cv. Arbequina was studied from November 1999 to May 2003 in a drip-irrigated, nontillage orchard established in a soil without a history of the disease at Córdoba, southern Spain. Disease incidence measured at 1-month-intervals increased from 0.2 to 7.8% during this period. Verticillium dahliae infecting the trees was characterized as defoliating (D) or nondefoliating (ND) pathotypes by a specific, multiplex-polymerase chain reaction (PCR) assay. Of the symptomatic trees, 87.2 and 12.8% were infected by the D or ND pathotypes, respectively. Dynamics of disease incidence were described by a generalized logistic model with a multiple sigmoid pattern. In the fitted model, the infection rate was highest in the winter to spring period and decreased to minimum values in the summer to fall period. Binary data of disease incidence was analyzed for point pattern and spatial correlation, either directly or after parsing them in contiguous quadrats. Overall, ordinary runs analysis indicated a departure from randomness of disease within rows. The binomial index of dispersion, interclass correlation, and Taylor's power law for various quadrat sizes suggested aggregation of diseased trees within the quadrat sizes tested. Spatial analysis by distance indices showed a nonrandom arrangement of quadrats containing infected trees. Spatial pattern was characterized by the occurrence of several clusters of infected trees. Increasing clustering over time was generally suggested by stronger values of clustering index over time and by the increase in the size of patch clusters. Significant spatial association was found in the clustering of diseased trees over time across cropping seasons; however, clustering was significant only for infections by D V. dahliae, indicating that infections by the D pathotype were aggregated around initial infections. The number and size of clusters of D V. dahliae-infected trees increased over time. Microsatellite-primed PCR assays of a representative number of V. dahliae isolates from diseased trees indicated that the majority of infecting D isolates shared the fingerprinting profile with D V. dahliae isolated from soil of a naturally infested cotton field in close proximity to the orchard, suggesting that short distance dispersal of the pathogen from this soil to the olive orchard may have occurred.

Abstract: Plants have evolved strategies of stimulating and supporting specific groups of antagonistic microorganisms in the rhizosphere as a defense against diseases caused by soilborne plant pathogens owing to a lack of genetic resistance to some of the most common and widespread soilborne pathogens. Some of the best examples of natural microbial defense of plant roots occur in disease suppressive soils. Soil suppressiveness against many different diseases has been described. Take-all is an important root disease of wheat, and soils become suppressive to take-all when wheat or barley is grown continuously in a field following a disease outbreak; this phenomenon is known as take-all decline (TAD). In Washington State, USA and The Netherlands, TAD results from the enrichment during monoculture of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens to a density of 10 (5) CFU/g of root, the threshold required to suppress the take-all pathogen, Gaeumannomyces graminis var. tritici. 2,4-DAPG-producing P. fluorescens also are enriched by monoculture of other crops such as pea and flax, and evidence is accumulating that 2,4-DAPG producers contribute to the defense of plant roots in many different agroecosystems. At this time, 22 distinct genotypes of 2,4-DAPG producers (designated A - T, PfY and PfZ) have been defined by whole-cell repetitive sequence-based (rep)-PCR analysis, restriction fragment length polymorphism (RFLP) analysis of PHLD, and phylogenetic analysis of PHLD, but the number of genotypes is expected to increase. The genotype of an isolate is predictive of its rhizosphere competence on wheat and pea. Multiple genotypes often occur in a single soil and the crop species grown modulates the outcome of the competition among these genotypes in the rhizosphere. 2,4-DAPG producers are highly effective biocontrol agents against a variety of plant diseases and ideally suited for serving as vectors for expressing other biocontrol traits in the rhizosphere.

Abstract: ABSTRACT Races 0 (Foc-0) and 5 (Foc-5) of Fusarium oxysporum f. sp. ciceris differ in virulence and induce yellowing or wilting syndrome, respectively, in chickpea. We modeled the combined effects of soil temperature and inoculum density of Foc-0 and Foc-5 on disease developed in chickpea cvs. P-2245 and PV-61 differing in susceptibility to those races, using quantitative nonlinear models. Disease development over time in the temperature range of 10 to 30 degrees C and inoculum densities between 6 and 8,000 chlamydospores g(1) of soil was described by the Weibull function. Four response variables (the reciprocal incubation period, the final disease intensity, the standardized area under the disease progress curve, and the intrinsic rate of disease development) characterized the disease development. Response surface models that expressed the combined effect of inoculum density and temperature were developed by substituting the intrinsic rate of disease development in the Weibull or exponential functions with a beta function describing the relationship of response variables to temperature. The models estimated 22 to 26 degrees C as the most favorable soil temperature for infection of cvs. P-2245 and PV-61 by Foc-5, and 24 to 28 degrees C for infection of cv. P-2245 by Foc-0. At 10 degrees C, no disease developed except in cv. P-2245 inoculated with Foc-5. At optimum soil temperature, maximum disease intensity developed with Foc-5 and Foc-0 at 6 and 50 chlamydospores g(1) of soil respectively, in cv. P-2245, and with Foc-5 at 1,000 chlamydospores g(1) of soil in cv. PV-61. The models were used to construct risk threshold charts that can be used to estimate the potential risk of Fusarium wilt epidemics in a geographical area based on soil temperature, the race and inoculum density in soil, and the level of susceptibility of the chickpea cultivar.

Abstract: ABSTRACT Severe downy mildew diseases of opium poppy (Papaver somniferum) can be caused by Peronospora arborescens and P. cristata, but differentiating between the two pathogens is difficult because they share morphological features and a similar host range. In Spain, where severe epidemics of downy mildew of opium poppy have occurred recently, the pathogen was identified as P. arborescens on the basis of morphological traits. In this current study, sequence homology and phylogenetic analyses of the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) were carried out with DNA from P. arborescens and P. cristata from diverse geographic origins, which suggested that only P. arborescens occurs in cultivated Papaver somniferum in Spain. Moreover, analyses of the rDNA ITS region from 27 samples of downy-mildew-affected tissues from all opium-poppy-growing regions in Spain showed that genetic diversity exists within P. arborescens populations in Spain and that these are phylogenetically distinct from P. cristata. P. cristata instead shares a more recent, common ancestor with a range of Peronospora species that includes those found on host plants that are not members of the Papaveraceae. Species-specific primers and a PCR assay protocol were developed that differentiated P. arborescens and P. cristata and proved useful for the detection of P. arborescens in symptomatic and asymptomatic opium poppy plant parts. Use of these primers demonstrated that P. arborescens can be transmitted in seeds and that commercial seed stocks collected from crops with high incidence of the disease were frequently infected. Field experiments conducted in microplots free from P. arborescens using seed stocks harvested from infected capsules further demonstrated that transmission from seedborne P. arborescens to opium poppy plants can occur. Therefore, the specific-PCR detection protocol developed in this study can be of use for epidemiological studies and diagnosing the pathogen in commercial seed stocks; thus facilitating the sanitary control of the disease and avoidance of the pathogen distribution in seeds.

Abstract: Races 0 (Foc-0) and 5 (Foc-5) of Fusarium oxysporum f. sp. ciceris differ in virulence and induce yellowing or wilting syndrome, respectively, in chickpea. We modeled the combined effects of soil temperature and inoculum density of Foc-0 and Foc-5 on disease developed in chickpea cvs. P-2245 and PV-61 differing in susceptibility to those races, using quantitative nonlinear models. Disease development over time in the temperature range of 10 to 30°C and inoculum densities between 6 and 8,000 chlamydospores g-1 of soil was described by the Weibull function. Four response variables (the reciprocal incubation period, the final disease intensity, the standardized area under the disease progress curve, and the intrinsic rate of disease development) characterized the disease development. Response surface models that expressed the combined effect of inoculum density and temperature were developed by substituting the intrinsic rate of disease development in the Weibull or exponential functions with a beta function describing the relationship of response variables to temperature. The models estimated 22 to 26°C as the most favorable soil temperature for infection of cvs. P-2245 and PV-61 by Foc-5, and 24 to 28°C for infection of cv. P-2245 by Foc-0. At 10°C, no disease developed except in cv. P-2245 inoculated with Foc-5. At optimum soil temperature, maximum disease intensity developed with Foc-5 and Foc-0 at 6 and 50 chlamydospores g-1 of soil respectively, in cv. P-2245, and with Foc-5 at 1,000 chlamydospores g-1 of soil in cv. PV-61. The models were used to construct risk threshold charts that can be used to estimate the potential risk of Fusarium wilt epidemics in a geographical area based on soil temperature, the race and inoculum density in soil, and the level of susceptibility of the chickpea cultivar. © 2007 The American Phytopathological Society.

Abstract: Beauveria bassiana strain EABb 04/01-Tip isolated from stem-borer larvae of Timaspis papaveris (Hymenoptera: Cynipidae), a serious pest of opium poppy in Spain, was shown to be able to become established endophytically in this pharmaceutical crop. Microbiological, molecular and light and electron microscopic methods were used to study fungal colonisation and to describe its mode of penetration. After inoculation with a foliar spray of conidia, microbiological methods showed 100% of plants examined 24, 48, 72 and 144 h after treatment to be colonised endophytically by the fungus, although the percentage of previously surface sterilised leaf pieces showing fungal growth was 100% at 24 and 48 h, and 80 and 75% at 72 and 144 h after treatment, respectively. The fungus was also observed in leaf pieces obtained from newly formed leaves, indicating that it could spread from treated leaves to leaves formed after fungal application. For molecular studies, a polymerase chain reaction (PCR) protocol was used to amplify the ITS1-5.8S-ITS2 regions of the rDNA of the plant and the fungus. This procedure allowed the detection of the fungus on the surface of the leaves and also endophytically, but only at 72 h after treatment. A nucleotide BLAST search revealed that the ITS1-5.8S-ITS2 sequence of strain EABb 04/01-Tip showed 100% homology with a similar sequence from Cordyceps bassiana. SEM images revealed that although numerous conidia were observed on the leaf surface, few germinated and penetrated. Intracellular colonisation by B. bassiana was not observed, but hyphae were detected growing into the xylem vessels. The fungus was found to colonise 40.5 +/- 4.3% of seedlings (with two cotyledons and the two first real leaves) from seeds dressed with a fungal spore suspension. These results may have implications in the biological control of T. papaveris, including the possible systemic protection of the plant against this cynipid.

Abstract: Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the suppressiveness of some soils to take-all of wheat and other diseases caused by soilborne pathogens. Soils from side-by-side fields on the campus of North Dakota State University, Fargo, USA, which have undergone continuous wheat, continuous flax or crop rotation for over 100 years, were assayed for the presence of 2,4-DAPG producers. Flax and wheat monoculture, but not crop rotation, enriched for 2,4-DAPG producers, and population sizes of log 5.0 CFU g root(-1) or higher were detected in the rhizospheres of wheat and flax grown in the two monoculture soils. The composition of the genotypes enriched by the two crops differed. Four BOX-PCR genotypes (D, F, G, and J) and a new genotype (T) were detected among the 2,4-DAPG producers in the continuous flax soil, with F- and J-genotype isolates dominating (41 and 39% of the total, respectively). In contrast, two genotypes (D and I) were detected in the soil with continuous wheat, with D-genotype isolates comprising 77% of the total. In the crop-rotation soil, populations of 2,4-DAPG producers generally were below the detection limit, and only one genotype (J) was detected. Under growth-chamber and field conditions, D and I genotypes (enriched by wheat monoculture) colonized the wheat rhizosphere significantly better than isolates of other genotypes, while a J-genotype isolate colonized wheat and flax rhizospheres to the same extent. This study suggests that, over many years of monoculture, the crop species grown in a field enriches for genotypes of 2,4-DAPG producers from the reservoir of genotypes naturally present in the soil that are especially adapted to colonizing the rhizosphere of the crop grown.

Abstract: Diversity within a worldwide collection of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains was assessed by sequencing the phlD gene. Phylogenetic analyses based on the phlD sequences of 70 isolates supported the previous classification into 18 BOX-PCR genotypes (A-Q and T). Exploiting polymorphisms within the sequence of phlD, we designed and used allele-specific PCR primers with a PCR-based dilution endpoint assay to quantify the population sizes of A-, B-, D-, K-, L- and P-genotype strains grown individually or in pairs in vitro, in the rhizosphere of wheat and in bulk soil. Except for P. fluorescens Q8r1-96, which strongly inhibited the growth of P. fluorescens Q2-87, inhibition between pairs of strains grown in vitro did not affect the accuracy of the method. The allele-specific primer-based technique is a rapid method for studies of the interactions between genotypes of 2,4-diacetylphloroglucinol producers in natural environments.

Abstract: ABSTRACT Strains of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are biocontrol agents which play a key role in the suppressiveness of some soils against soilborne pathogens. We evaluated the effect of the host plant genotype on rhizosphere colonization by both indigenous and introduced 2,4-DAPG-producing P. fluorescens. First, population densities of indigenous 2,4-DAPG producers in the rhizospheres of alfalfa, barley, bean, flax, lentil, lupine, oat, pea, and wheat grown in a Fusarium wilt-suppressive Puget silt loam were determined. Population densities differed among the various crops and among pea cultivars, with lentil and oat supporting the highest and lowest densities of 2,4-DAPG producers, respectively. Second, to determine the interactions among 2,4-DAPG producers in the rhizosphere, a Shano sandy loam was inoculated individually and with all possible combinations of P. fluorescens Q8r1-96 (genotype D), F113 (genotype K), and MVP1-4 (genotype P) and sown to wheat or pea, and the rhizosphere population dynamics of each strain was monitored. All three strains were similar in ability to colonize the rhizosphere of wheat and pea when introduced alone into the soil; however, when introduced together in equal densities, the outcome of the interactions differed according to the host crop. In the wheat rhizosphere, the population density of strain F113 was significantly greater than that of Q8r1-96 in the mixed inoculation studies, but no significant differences were observed on pea. The population density of strain Q8r1-96 was greater than that of MVP1-4 in the mixed inoculation on wheat, but the opposite occurred on pea. In the wheat rhizosphere, the population of MVP1-4 dropped below the detection limit (log 3.26 CFU g(-1) of root) in the presence of F113; however, on pea, the population density of MVP1-4 was higher than that of F113. When all three strains were present together, F113 had the greatest density in the wheat rhizosphere, but MVP1-4 was dominant in the pea rhizosphere. Finally, eight pea cultivars were grown in soil inoculated with either MVP1-4 or Q8r1-96. The effect of the pea cultivar on rhizosphere colonization was dependent on the bacterial strain inoculated. Rhizosphere population densities of MVP1-4 did not differ significantly among pea cultivars, whereas population densities of Q8r1-96 did. We conclude from these studies that the host crop plays a key role in modulating both rhizosphere colonization by 2,4-DAPG-producing P. fluorescens and the interactions among different genotypes present in the same rhizosphere.

Abstract: Use of resistant cultivars and adjustment of sowing dates are important measures for management of Fusarium wilt in chickpeas (Cicer arietinum). In this study, we examined the effect of temperature on resistance of chickpea cultivars to Fusarium wilt caused by various races of Fusarium oxysporum f. sp. ciceris. Greenhouse experiments indicated that the chickpea cultivar Ayala was moderately resistant to F. oxysporum f. sp. ciceris when inoculated plants were maintained at a day/night temperature regime of 24/21°C but was highly susceptible to the pathogen at 27/25°C. Field experiments in Israel over three consecutive years indicated that the high level of resistance of Ayala to Fusarium wilt when sown in mid- to late January differed from a moderately susceptible reaction under warmer temperatures when sowing was delayed to late February or early March. Experiments in growth chambers showed that a temperature increase of 3°C from 24 to 27°C was sufficient for the resistance reaction of cultivars Ayala and PV-1 to race 1A of the pathogen to shift from moderately or highly resistant at constant 24°C to highly susceptible at 27°C. A similar but less pronounced effect was found when Ayala plants were inoculated with F. oxysporum f. sp. ciceris race 6. Conversely, the reaction of cultivar JG-62 to races 1A and 6 was not influenced by temperature, but less disease developed on JG-62 plants inoculated with a variant of race 5 of F. oxysporum f. sp. ciceris at 27°C compared with plants inoculated at 24°C. These results indicate the importance of appropriate adjustment of temperature in tests for characterizing the resistance reactions of chickpea cultivars to the pathogen, as well as when determining the races of isolates of F. oxysporum f. sp. ciceris. Results from this study may influence choice of sowing date and use of chickpea cultivars for management of Fusarium wilt of chickpea. © 2006 The American Phytopathological Society.

Abstract: A 3-year experiment was conducted in field microplots infested with Fusarium oxysporum f. sp. ciceris race 5 at Co?rdoba, Spain, in order to assess efficacy of an integrated management strategy for Fusarium wilt of chickpea that combined the choice of sowing date, use of partially resistant chickpea genotypes, and seed and soil treatments with biocontrol agents Bacillus megaterium RGAF 51, B. subtilis GB03, nonpathogenic F. oxysporum Fo 90105, and Pseudomonas fluorescens RG 26. Advancing the sowing date from early spring to winter significantly delayed disease onset, reduced the final disease intensity (amount of disease in a microplot that combines disease incidence and severity, expressed as a percentage of the maximum possible amount of disease in that microplot), and increased chickpea seed yield. A significant linear relationship was found between disease development over time and weather variables at the experimental site, with epidemics developing earlier and faster as mean temperature increased and accumulated rainfall decreased. Under conditions highly conducive for Fusarium wilt development, the degree of disease control depended primarily on choice of sowing date, and to a lesser extent on level of resistance of chickpea genotypes to F. oxysporum f. sp. ciceris race 5, and the biocontrol treatments. The main effects of sowing date, partially resistant genotypes, and biocontrol agents were a reduction in the rate of epidemic development over time, a reduction of disease intensity, and an increase in chickpea seedling emergence, respectively. Chickpea seed yield was influenced by all three factors in the study. The increase in chickpea seed yield was the most consistent effect of the biocontrol agents. However, that effect was primarily influenced by sowing date, which also determined disease development. Effectiveness of biocontrol treatments in disease management was lowest in January sowings, which were least favorable for Fusarium wilt. Sowing in February, which was moderately favorable for wilt development, resulted in the greatest increase in seed yield by the biocontrol agents. In March sowings, which were most conducive for the disease, the biocontrol agents delayed disease onset and increased seedling emergence. B. subtilis GB03 and P. fluorescens RG 26, applied either alone or each in combination with nonpathogenic F. oxysporum Fo 90105, were the most effective treatments at suppressing Fusarium wilt, or delaying disease onset and increasing seed yield, respectively. The importance of integrating existing control practices, partially effective by themselves, with other control measures to achieve appropriate management of Fusarium wilt and increase of seed yield in chickpea in Mediterranean-type environments is demonstrated by the results of this study.

Abstract: Seed and soil treatment with Pseudomonas fluorescens RGAF 19, P. fluorescent RG 26, Bacillus megaterium RGAF 51 and Paenibacillus macerans RGAF 101 can suppress fusarium wilt of chickpea (Cicer arietinum), but the extent of disease suppression by these rhizobacteria is modulated by soil temperature. In this work, the effect of temperature on plant-rhizobacteria interactions was assessed in relation to biocontrol potential for suppression of fusarium wilt of chickpea. Seed and soil treatment with those rhizobacteria delayed seedling emergence compared with nontreated controls, and either increased or had no deleterious effect on chickpea growth. Pseudomonas fluorescens isolates significantly increased chickpea shoot dry weight at 20°C and root dry weight at 25 and 30°C. All bacterial isolates colonized the chickpea rhizosphere and internal stem tissues at 20, 25 and 30°C, and there was a positive linear trend between bacterial population size in the rhizosphere and temperature increase. The maximum inhibition of mycelial growth and conidial germination of Fusarium oxysporum f. sp. ciceris race 5 in vitro occurred at a temperature range optimal for bacterial growth and production of inhibitory metabolites. These results demonstrate the need to understand the effects of environmental factors on the biological activities of introduced rhizobacteria of significant importance for plant disease suppression.

Abstract: ABSTRACT A 3-year experiment was conducted in field microplots infested with Fusarium oxysporum f. sp. ciceris race 5 at Córdoba, Spain, in order to assess efficacy of an integrated management strategy for Fusarium wilt of chickpea that combined the choice of sowing date, use of partially resistant chickpea genotypes, and seed and soil treatments with biocontrol agents Bacillus megaterium RGAF 51, B. subtilis GB03, nonpathogenic F. oxysporum Fo 90105, and Pseudomonas fluorescens RG 26. Advancing the sowing date from early spring to winter significantly delayed disease onset, reduced the final disease intensity (amount of disease in a microplot that combines disease incidence and severity, expressed as a percentage of the maximum possible amount of disease in that microplot), and increased chickpea seed yield. A significant linear relationship was found between disease development over time and weather variables at the experimental site, with epidemics developing earlier and faster as mean temperature increased and accumulated rainfall decreased. Under conditions highly conducive for Fusarium wilt development, the degree of disease control depended primarily on choice of sowing date, and to a lesser extent on level of resistance of chickpea genotypes to F. oxysporum f. sp. ciceris race 5, and the biocontrol treatments. The main effects of sowing date, partially resistant genotypes, and biocontrol agents were a reduction in the rate of epidemic development over time, a reduction of disease intensity, and an increase in chickpea seedling emergence, respectively. Chickpea seed yield was influenced by all three factors in the study. The increase in chickpea seed yield was the most consistent effect of the biocontrol agents. However, that effect was primarily influenced by sowing date, which also determined disease development. Effectiveness of biocontrol treatments in disease management was lowest in January sowings, which were least favorable for Fusarium wilt. Sowing in February, which was moderately favorable for wilt development, resulted in the greatest increase in seed yield by the biocontrol agents. In March sowings, which were most conducive for the disease, the biocontrol agents delayed disease onset and increased seedling emergence. B. subtilis GB03 and P. fluorescens RG 26, applied either alone or each in combination with nonpathogenic F. oxysporum Fo 90105, were the most effective treatments at suppressing Fusarium wilt, or delaying disease onset and increasing seed yield, respectively. The importance of integrating existing control practices, partially effective by themselves, with other control measures to achieve appropriate management of Fusarium wilt and increase of seed yield in chickpea in Mediterranean-type environments is demonstrated by the results of this study.

Abstract: ABSTRACT Strains of fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphoroglucinol (2,4-DAPG) are among the most effective rhizobacteria controlling diseases caused by soilborne pathogens. The genotypic diversity that exists among 2,4-DAPG producers can be exploited to improve rhizosphere competence and biocontrol activity. Knowing that D-genotype 2,4-DAPG-producing strains are enriched in some take-all decline soils and that P. fluorescens Q8r1-96, a representative D-genotype strain, as defined by whole-cell repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer, is a superior colonizer of wheat roots, we analyzed whether the exceptional rhizosphere competence of strain Q8r1-96 on wheat is characteristic of other D-genotype isolates. The rhizosphere population densities of four D-genotype strains and a K-genotype strain introduced individually into the soil were significantly greater than the densities of four strains belonging to other genotypes (A, B, and L) and remained above log 6.8 CFU/g of root over a 30-week cycling experiment in which wheat was grown for 10 successive cycles of 3 weeks each. We also explored the competitive interactions between strains of different genotypes inhabiting the same soil or rhizosphere when coinoculated into the soil. Strain Q8r1-96 became dominant in the rhizosphere and in nonrhizosphere soil during a 15-week cycling experiment when mixed in a 1:1 ratio with either strain Pf-5 (A genotype), Q2-87 (B genotype), or 1M1-96 (L genotype). Furthermore, the use of the de Wit replacement series demonstrated a competitive disadvantage for strain Q2-87 or strong antagonism by strain Q8r1-96 against Q2-87 in the wheat rhizosphere. Amplified rDNA restriction analysis and sequence analysis of 16S rDNA showed that species of Arthrobacter, Chryseobacterium, Flavobacterium, Massilia, Microbacterium, and Ralstonia also were enriched in culturable populations from the rhizosphere of wheat at the end of a 30-week cycling experiment in the presence of 2,4-DAPG producers. Identifying the interactions among 2,4-DAPG producers and with other indigenous bacteria in the wheat rhizosphere will help to elucidate the variability in biocontrol efficacy of introduced 2,4-DAPG producers and fluctuations in the robustness of take-all suppressive soils.

Abstract: Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)(-1) over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.

Abstract: Suppression of soilborne diseases by biocontrol agents involves complex interactions among biocontrol agents and the pathogen and between these microorganisms and the plant. In general, these interactions are not well characterized. In this work, we studied (i) the diversity among strains of fluorescent Pseudomonas spp., Bacillus spp., and Paenibacillus sp. for their sensitivity to fusaric acid (FAc) and phytoanticipins from different host plants, (ii) the diversity of pathogenic and nonpathogenic Fusarium oxysporum isolates for their sensitivity to phytoanticipins, and (iii) the influence of FAc on the production of pyoverdine by fluorescent Pseudomonas spp. tolerant to this compound. There was a great diversity in the response of the bacterial strains to FAc; however, as a group, Bacillus spp. and Paenibacillus macerans were much more sensitive to FAc than Pseudomonas spp. FAc also affected production of pyoverdine by FAc-tolerant Pseudomonas spp. strains. Phytoanticipins differed in their effects on microbial growth, and sensitivity to a phytoanticipin varied among bacterial and fungal strains. Biochanin A did not affect growth of bacteria, but coumarin inhibited growth of Pseudomonas spp. strains and had no effect on Bacillus circulans and P. macerans. Conversely, tomatine inhibited growth of B. circulans and P. macerans. Biochanin A and tomatine inhibited growth of three pathogenic isolates of F. oxysporum but increased growth of three nonpathogenic F. oxysporum isolates. Coumarin inhibited growth of all pathogenic and nonpathogenic F. oxysporum isolates. These results are indicative of the complex interactions that can occur among plants, pathogens, and biological control agents in the rhizosphere and on the root surface. Also, these results may help to explain the low efficacy of some combinations of biocontrol agents, as well as the inconsistency in achieving disease suppression under field conditions.

Abstract: Certain 2,4-diacetylphloroglucinol-producing strains of Pseudomonas fluorescens colonize roots and suppress soilborne diseases more effectively than others from which they are otherwise phenotypically almost indistinguishable. We recovered DNA fragments present in the superior colonizer P. fluorescens Q8r1-96 but not in the less rhizosphere-competent strain Q2-87. Of the open reading frames in 32 independent Q8r1-96-specific clones, 1 was similar to colicin M from Escherichia coli, 3 resembled known regulatory proteins, and 28 had no significant match with sequences of known function. Seven clones hybridized preferentially to DNA from strains with superior rhizosphere competence, and sequences in two others were highly expressed in vitro and in the rhizosphere.

Abstract: ABSTRACT Pseudomonas fluorescens strains producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) have biocontrol activity against a broad spectrum of root and seedling diseases. In this study, we determined the effect of genotype on the ability to isolate and quantify introduced 2,4-DAPG producers from the rhizosphere of wheat using three different methods: traditional dilution plating on selective media, colony hybridization followed by polymerase chain reaction (PCR), and phlD-specific PCR-based dilution endpoint assay. Regression analysis of the population densities of 10 2,4-DAPG-producing P. fluorescens, representing five genotypes, determined by the three different methods demonstrated that the relationship was linear (P < 0.001) and the techniques were very similar (i.e., slopes equal to 1.0). The phlD-specific PCR-based assay had a slightly lower limit of detection than the other two methods (log 3.3 versus log 4.0 CFU/g of fresh root weight). With the colony hybridization procedure, we observed that the phlD probe, derived from strain P. fluorescens Q8r1-96, hybridized more strongly to colonies of BOX-PCR genotypes D (strains W2-6, L5.1-96, Q8r1-96, and Q8r2-96) and K (strain F113) compared with strains of genotypes A (Pf-5 and CHA0), B (Q2-87), and L (1M1-96 and W4-4). Colony hybridization alone overestimated the actual densities of some strains, thus requiring an additional PCR step to obtain accurate estimates. In contrast, population densities estimated for three of the bacterial treatments (strains CHA0, W2-6, and Q8r2-96) with the PCR-based assay were significantly (P < 0.041) smaller by 7.6 to 9.2% and 6.4 to 9.4% than population densities detected by the dilution plating and colony hybridization techniques, respectively. In this paper, we discuss the relative advantages of the different methods for detecting 2,4-DAPG producers.

Abstract: The effects of temperature and inoculum density of Fusarium oxysporum f. sp. ciceris race 5 on suppression of Fusarium wilt in chickpea (Cicer arietinum) cv. PV 61 by seed and soil treatments with rhizobacteria isolated from the chickpea rhizosphere were studied in a model system. Disease development over a range of temperatures (20, 25, and 30 degrees C) and inoculum densities (25 to 1,000 chlamydospores per gram of soil) was described by the Gompertz model. The Gompertz relative rate of disease progress and final amount of disease increased exponentially and monomolecularly, respectively, with increasing inoculum densities. Disease development was greater at 25 degrees C compared with 20 and 30 degrees C. At 20 and 30 degrees C, disease development was greater at 250 to 1,000 chlamydospores per gram of soil compared with 25 to 100 chlamydospores per gram of soil. At 25 degrees C, increasing inoculum densities of the pathogen did not influence disease. Nineteen Bacillus, Paenibacillus, Pseudomonas, and Stenotrophomonas spp. out of 23 bacterial isolates tested inhibited F. oxysporum f. sp. ciceris in vitro. Pseudomonas fluorescens RGAF 19 and RG 26, which did not inhibit the pathogen, showed the greatest Fusarium wilt suppression. Disease was suppressed only at 20 or 30 degrees C and at inoculum densities below 250 chlamydospores per gram of soil. Bacterial treatments increased the time to initial symptoms, reduced the Gompertz relative rate of disease progress, and reduced the overall amount of disease developed.

Abstract: The effects of temperature and inoculum density of Fusarium oxysporum f. sp. ciceris race 5 on suppression of Fusarium wilt in chickpea (Cicer arietinum) cv. PV 61 by seed and soil treatments with rhizobacteria isolated from the chickpea rhizosphere were studied in a model system. Disease development over a range of temperatures (20, 25, and 30°C) and inoculum densities (25 to 1,000 chlamydospores per gram of soil) was described by the Gompertz model. The Gompertz relative rate of disease progress and final amount of disease increased exponentially and monomolecularly, respectively, with increasing inoculum densities. Disease development was greater at 25°C compared with 20 and 30°C. At 20 and 30°C, disease development was greater at 250 to 1,000 chlamydospores per gram of soil compared with 25 to 100 chiamydospores per gram of soil. At 25°C, increasing inoculum densities of the pathogen did not influence disease. Nineteen Bacillus, Paenibacillus, Pseudomonas, and Stenotrophomonas spp. out of 23 bacterial isolates tested inhibited F. oxysporum f. sp. ciceris in vitro. Pseudomonas fluorescens RGAF 19 and RG 26, which did not inhibit the pathogen, showed the greatest Fusarium wilt suppression. Disease was suppressed only at 20 or 30°C and at inoculum densities below 250 chlamydospores per gram of soil. Bacterial treatments increased the time to initial symptoms, reduced the Gompertz relative rate of disease progress, and reduced the overall amount of disease developed.