Improvement of seed bio-priming of oilseed rape (Brassica napus ssp. oleifera) with Serratia plymuthica and Pseudomonas chlororaphis

Seed bio-priming of oilseed rape (Brassica napus) with the antagonistic rhizobacteria Serratia plymuthica and Pseudomonas chlororaphis was improved. With the imbibition of water, bacteria are transported into the seed where they survive better. To obtain a minimum bacterial density in the seed of log₁₀ 5 colony-forming-units (CFUs) seed^?1, the bacterial density in the bio-priming suspension should be >log₁₀ 9 CFUs mL^–1 for S. plymuthica and >log₁₀ 8 CFUs mL^–1 for P. chlororaphis. Priming duration was reduced from 12 to 2 h for S. plymuthica and 4 h for P. chlororaphis. Among other priming solutions tested, the addition of MgSO₄ best supported establishment in the seeds and also improved germination. The optimal bio-priming temperature for S. plymuthica is 28°C and for P. chlororaphis 22°C. Survival of the bacteria inside the seeds was moderately improved by storage at low temperature but considerably prolonged by storage under anaerobic conditions. P. chlororaphis survived significantly longer than S. plymuthica.     

Molecular Characterization of the Host-Adapted Pathogen Verticillium longisporum on the Basis of a Group-I Intron Found in the Nuclear SSU-rRNA Gene

Verticillium wilt of oilseed rape is caused by the host-adapted pathogen Verticillium longisporum comb. nov. With one set of nuclear SSU-rRNA gene primers, a PCR amplification product of ca. 2.5 kb was generated from all isolates of V. longisporum tested (36 from Europe, Japan, and USA), with the exception of two recombinant isolates. On the contrary, all the other phytopathogenic and non-phytopathogenic species of Verticillium tested (18 species, 46 isolates), with the exception of one isolate of V. lecanii and two of Cordyceps sp., generated a product of ca. 1.65 kb. Sequence analysis of the SSU-rRNA gene of two typical isolates of V. longisporum (wild radish, Japan, and oilseed rape, Germany) revealed that this dimorphism was due to the presence of an identical 839-bp intron located in a highly conserved insertion position (nt 1165 of Saccharomyces cerevisiae). The intron sequence was classified as group-I intron on the basis of conserved sequence and secondary structural elements. Primers designed from the 839-bp intron sequence amplified only the V. longisporum. Phylogenetic analysis based on SSU-rDNA sequences showed that V. longisporum was closely related to the genera of other filamentous Ascomycetes with fruiting bodies. Received: 24 August 2000 / Accepted: 25 September 2000     

Impact of formulation procedures on the effect of the biocontrol agent Serratia plymuthica HRO-C48 on Verticillium wilt in oilseed rape

Verticillium wilt is an important disease in oilseed rape with an increasing importance worldwide. Currently, there are no methods available to suppress the pathogen. A biological protection strategy on the basis of the plant-beneficial bacterium Serratia plymuthica HRO-C48 to control Verticillium dahliae in oilseed rape was developed. Three different techniques to apply the biocontrol agent to seeds, namely pelleting, film coating and bio-priming, were evaluated considering the influence on the control activity, cell stability during storage and practical feasibility. Neither the treatment nor the inoculum density was found to influence the abundances of HRO-C48 in the rhizosphere after 30 days. Serratia treatment using bio-priming and pelleting resulted in a statistically significant biocontrol in comparison to the non-bacterized controls. Additionally, survival of HRO-C48 differed between treatments, and was the highest using bio-priming at 20°C, and pelleting at 4°C. In conclusion, the procedure of bio-priming, which was developed in line with this study, resulted in a stable and efficient formulation of S. plymuthica on rape seed. This technology opens a possibility to develop a commercial Serratia formulation to protect oilseed against V. dahliae.     

Genetic analysis of phenylpropanoid metabolites associated with resistance against Verticillium longisporum in Brassica napus

Verticillium longisporum is a major threat to production of oilseed rape (Brassica napus) in Europe. The aim of the study was to develop new markers and obtain insights into putative mechanisms and pathways involved in the resistance reaction. A genetic approach was used to identify quantitative trait loci (QTL) for V. longisporum resistance and metabolic traits potentially influencing resistance in a B. napus mapping population. Resistance to V. longisporum was mapped in a doubled haploid (DH) population from a cross between the partially resistant winter oilseed rape variety Express 617 and a resistant resynthesized B. napus line, R53. One major resistance QTL contributed by R53 was identified on chromosome C5, while a further, minor QTL contributed by Express 617 was detected on chromosome C1. Markers flanking the QTL also significantly correlated with V. longisporum resistance in four further DH populations derived from crosses between elite oilseed rape cultivars and other resynthesized B. napus lines originating from genetically and geographically diverse brassica A and C genome donors. The tightly-linked markers developed enable the combination of favorable alleles for novel resistance loci from resynthesized B. napus materials with existing resistance loci from commercial breeding lines. HPLC analysis of hypocotyls from infected DH lines revealed that concentrations of a number of phenylpropanoids were correlated with V. longisporum resistance. QTL for some of these phenylpropanoids were also found to co-localize with the QTL for V. longisporum resistance. Genes from the phenylpropanoid pathway are suggested as candidates for V. longisporum resistance.     

Plant-Dependent Genotypic and Phenotypic Diversity of Antagonistic Rhizobacteria Isolated from Different Verticillium Host Plants

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards Verticillium. The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.     

The role of antibiosis and induced systemic resistance, mediated by strains of Pseudomonas chlororaphis, Bacillus cereus and B. amyloliquefaciens, in controlling blackleg disease of canola

Antibiotic-producing Pseudomonas chlororaphis strains DF190 and PA23, Bacillus cereus strain DFE4 and Bacillus amyloliquefaciens strain DFE16 were tested for elicitation of induced systemic resistance (ISR) and direct antibiosis in control of blackleg in canola caused by the fungal pathogen Leptosphaeria maculans. Inoculation of bacteria 24 h and 48 h prior to the pathogen was crucial for disease control. In systemic induction studies, the bacteria and culture extracts had lower but significant suppression of the blackleg lesion. When inoculated at the same wound site as the pathogen pycnidiospores, the bacterial culture extracts had significantly higher reduction of blackleg lesion development. However, localized plant defense-related enzyme activity at the site of inoculation was not induced by all the bacteria. Direct antifungal activity at the infection site seems to be the dominant mechanism mediating control of L. maculans. A Tn5-gacS mutant of strain PA23 exhibited a complete loss of antifungal and biocontrol activity, which was restored upon addition of the gacS gene in trans. Interestingly, a phenazine-minus derivative of PA23 that produces elevated levels of pyrrolnitrin exhibited the same or higher blackleg disease suppression compared to the wild type. These findings suggest that direct antifungal activity, possibly mediated by pyrrolnitrin, and some low level of induced systemic resistance is involved in P. chlororaphis biocontrol of blackleg.     

Characteristics of Bacteria from Oilseed Rape in Relation to their Biocontrol Activity against Verticillium dahliae

The potential of bacteria that are adapted to the oilseed rape root environment for use in the biological control of Verticillium dahliae, Kleb was investigated in both controlled and non‐sterile growth conditions. Bacterial strains dominated by the red‐pigmented members of enterobacteriaceae were isolated from thoroughly washed and air‐dried root segments of symptomless young rape plants. Other associated strains found either belonged to Alcaligenes sp., Stenotrophomonas spp. and Pseudomonas spp. (Pseudomonas acidovorans and Pseudomonas putida) or were unidentified according to fatty acid methyl ester profile analysis. A total of 19 strains isolated in this study together with two previously studied strains, Serratia proteamaculans and Pseudomonas chlororaphis, were characterized on the basis of their interactions with V. dahliae and a number of functional characteristics. In line with earlier observations with root‐colonizing fungi also from oilseed rape, all bacterial strains suppressed the pathogen not only directly and but also indirectly in in vitro assays. Mechanisms of suppression were apparently multifold among the strains, but production of hydrogen cyanide does not seem to be involved in indirect inhibition. The majority of the strains possessed the ability to produce cellulases, proteases and phosphatases and some even produced chitinases and induced hypersensitive responses, indicating their potential for nutrient acquisition as well as colonization capacity and active recognition by the plant cells. Investigations in non‐sterile field soil revealed that some strains protected rape plants from V. dahliae partly by delaying symptom development. None of the strains, however, was strongly deleterious to rape growth either in the presence or absence of the pathogen. Light microscopic observations of roots and results based on agar printing techniques revealed the potential of the studied strains to colonize or interfere with the pathogen colonization. This study provides some insight into the evolved relationship of bacterial residents with their host in terms of their potential importance in its fitness.     

Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.     

水产动物副溶血性弧菌的拮抗菌研究进展

细菌性疾病的爆发常造成水产养殖业的巨大经济损失,其中副溶血性弧菌(Vibrio parahaemolyticus)引起的细菌性疾病更是引起了人们的关注.拮抗菌在代谢活动中通过分泌抗菌物质直接对病原菌产生抑制或竞争作用来抑制或杀死病原菌,在病害防治中发挥着重大作用.对副溶血性弧菌拮抗菌的种类、产生拮抗物质的种类、筛选拮抗...     

黑胫病菌侵染过程中油菜响应基因的表达分析

油菜黑胫病是造成油菜产量损失的病害之一,致病菌为Leptosphaeria biglobosa.该研究采用形态学观察和转录组测序技术,分析油菜接种病原菌Leptosphaeria biglobosa 4、12、24、36、48和96 h后的表型及基因表达变化情况,以探讨响应死体营养型真菌L.biglobosa侵染时油菜...     

Biocontrol potential of Microsphaeropsis ochracea on microsclerotia of Verticillium longisporum in environments differing in microbial complexity

The potential of the fungal antagonist Microsphaeropsis ochracea to control the soilborne pathogen Verticillium longisporum was investigated in environments with varying microbial complexity (in vitro vs. in vivo, sterile vs. unsterile, controlled conditions vs. field). A semi-quantitative PCR assay was developed for the detection of M. ochracea on unsterile plant debris. In vitro, M. ochracea caused high levels of mortality to V. longisporum microsclerotia (51–100 %) from 4 to 24 °C, with a broad optimum between 16 and 24 °C. In controlled conditions, M. ochracea significantly reduced the viability of V. longisporum microsclerotia grown on dead rapeseed stems in autoclaved sand, but not in unsterile soil. Likewise, in two experimental years, no significant reduction of V. longisporum inoculum was detectable on rapeseed straw buried in small plots in the field in any of the treatments (soil depths, exposure duration, doses of M. ochracea). Germination of M. ochracea pycnidiospores was inhibited by general soil fungistasis in unsterile soil from a field, botanical garden and grassland. Accordingly, V. longisporum infection of rapeseed plants in the greenhouse was reduced only at artificially high doses of M. ochracea inoculum and no biocontrol efficacy in disease control was recorded in field experiments conducted with winter oilseed rape during two subsequent seasons in an experimental field near Göttingen, with a soil homogenously infested with V. longisporum. The results demonstrate that M. ochracea, although having shown promising potential in controlling pathogens with melanised resting structures on leaf litter, evidently lacks microbial competitiveness to effectively control pathogens in the soil such as V. longisporum, even though the latter is effectively inhibited in vitro.     

Application of RAPD-PCR for Virulence Type Analysis within Verticillium dahliae and V. longisporum

A collection of 24 isolates of Verticillium dahliae, 11 isolates of V. longisporum and one isolate of V. albo‐atrum originating from different host plants and geographical regions was tested for genetic variability by random amplified polymorphic DNA‐polymerase chain reaction (RAPD–PCR). Based on nine primers, the three Verticillium species could be clearly differentiated. Likewise, this analysis provided a distinct separation of vegetative compatibility groups (VCG) 2B, 4A and 4B of V. dahliae by specific DNA banding patterns. Additionally, V. longisporum was found to segregate into two subgroups with only 88% similarity. This molecular‐genetic approach was used for the analysis of randomly selected Verticillium isolates from a field with high intensity of oilseed rape cultivation (33% in crop rotation). RAPD‐PCR analysis revealed that 95 of 100 isolates tested belonged to V. longisporum and 5 to VCG 2B of V. dahliae. This study demonstrates an adaptation of Verticillium soil populations to a specific cropping history.     

Effects of experimental warming on fungal disease progress in oilseed rape

Global warming will influence the growth and development of both crops and pathogens. The aims of this study were to investigate potential effects of future warming on oilseed rape growth and the epidemiology of the three economically important pathogens Verticillium longisporum, Sclerotinia sclerotiorum, and Leptosphaeria maculans (anamorph: Phoma lingam). We utilized climate chambers and a soil warming facility, where treatments represented regional warming scenarios for Lower Saxony, Germany, by 2050 and 2100, and compared results of both approaches on a thermal time scale by calculating degree‐days (dd) from day of sowing, December 1st and March 1st until sampling, the latter correlating best with disease progress. Regression analysis showed that plant growth and growth stages in spring responded almost linearly to increasing thermal time until 1000–1500 dd. Colonization of plant tissue by V. longisporum showed an exponential increase when exceeding 1300–1500 dd and reaching plant growth stage BBCH 74/75 (pod development). V. longisporum colonization of plants may be advanced, potentially leading to higher inoculum densities after harvest and increased economic importance of this pathogen under future warming. Sclerotia germination of S. sclerotiorum reached its maximum at 600–900 dd. Advance of these critical degree‐days may lead to earlier apothecia production, potentially advancing the infection window, whereas the future importance of S. sclerotiorum may remain constant. Severity of phoma crown canker increased linearly with increasing thermal time, but showed also large variation in response to the warming scenarios, suggesting that factors such as canopy microclimate in fall or leaf shedding over winter may play a bigger role for L. maculans infection and disease severity than higher soil temperatures. Thermal time was a suitable tool to combine and integrate data on biological responses to soil and air temperature increases from climate chamber and field experiments.     

Plant growth-promotion (PGP) activities and molecular characterization of rhizobacterial strains isolated from soybean (Glycine max L. Merril) plants against charcoal rot pathogen, Macrophomina phaseolina

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, leads to significant yield losses of soybean crops. One strategy to control charcoal rot is the use of antagonistic, root-colonizing bacteria. Rhizobacteria A₅F and FPT₇21 and Pseudomonas sp. strain GRP₃ were characterized for their plant growth-promotion activities against the pathogen. Rhizobacterium FPT₇21 exhibited higher antagonistic activity against the pathogen on dual plate assay compared to strain A₅F and GRP₃. FPT₇21 and GRP₃ gave decreased disease intensity in terms of average number of pathogen-infested plants. Lipoxygenase (LOX), phenylalanine ammonia-lyase (PAL), and peroxidase (POD) activities were estimated in extracts of plants grown from seeds that were treated with rhizobacteria, and inoculated with spore suspension of M. phaseolina. The activity of these enzymes after challenge with the test pathogen increased. Strains FPT₇21 and GRP₃ exhibited maximum increases in LOX, PAL and POD activity (U mg⁻¹ fresh leaf wt) compared to strain A₅F.     

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO₂ and H₂O₂. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H₂O₂ levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum. Xiangbai Dong and Ruiqin Ji contributed equally to this paper.     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.     

Identification of Cauliflower Cultivars that Differ in Susceptibility to Verticillium longisporum using Different Inoculation Methods

The response of 13 European cauliflower cultivars to Verticillium longisporum was evaluated using two greenhouse tests and one in vitro inoculation test. The greenhouse tests involved dipping roots of 3‐week‐old seedlings in a conidial suspension or inoculating the soil of 3‐week‐old seedlings with Verticillium microsclerotia. The in vitro test involved the inoculation of 9‐day‐old seedlings with Verticillium conidia. Useful disease parameters were the area under disease progress curve and plant growth reduction for the greenhouse tests and fresh weight reduction for the in vitro test. Significant correlations were found among the three inoculation methods. Irrespective of the inoculation method used, cultivar ‘Sernio’ was most resistant to V. longisporum, while ‘Minaret’ was the most susceptible cultivar. The pathogen could be re‐isolated from the hypocotyls and from the stem of ‘Minaret’ 4 and 49 days after inoculation respectively, whereas V. longisporum could never be re‐isolated from ‘Sernio’. These results suggest that the more resistant cauliflower cultivar ‘Sernio’ can suppress the ascent and the proliferation of V. longisporum into the plant.     

Pre-germinated conidia of Coniothyrium minitans enhances the foliar biological control of Sclerotinia sclerotiorum

The relatively slow germination rate of Coniothyrium minitans limits its control efficiency against Sclerotinia sclerotiorum. Pre-germinated conidia of C. minitans enhanced its efficiency significantly: in foliar experiments with oilseed rape, hyphal extension of S. sclerotiorum was inhibited by 68%, while formation of sclerotia was completely inhibited when pre-germinated conidia were applied.Revisions requested 27 July 2004; Revisions received 7 September