Postharvest Biological Control of Potato Sprouting by Fusarium Dry Rot Suppressive Bacteria

Chemical sprout inhibitors are applied to over 50% of the potato harvest to extend storage time. In the U.S.A., CIPC (1-methylethyl-3-chlorophenylcarbamate) is the only synthetic chemical registered for postharvest sprout control of stored potatoes, and it is the most widely used sprout inhibitor world-wide. Due to environmental and health safety concerns, the use of CIPC has become more restricted, and alternative sprout control methods are sought. Six bacteria strains, exhibiting superior dry rot suppressiveness in previous research, were grown in two different liquid culture media and sprayed on Russet Burbank potatoes. In growth chamber and pilot experiments repeated at two storage sites in two successive years, all six isolates demonstrated significant sprout control capabilities when applied after growth on at least one of the culture media supplied. Of the six strains tested, Pseudomonas fluorescens bv.V S11:P:12 and two strains of Enterobacter sp., S11:T:07 and S11:P:08, exhibited highest relative performance levels with sprout control being statistically similar to that of 16.6 ppm CIPC thermal fog after 4-5 months storage.     

Hydrolytic Activities of Fusarium solani and Fusarium solani f. sp. eumartii Associated with the Infection Process of Potato Tubers

The development of dry rot caused by Fusarium solani f. sp. eumartii was evaluated in susceptible (Huinkul) and resistant (Spunta) potato cultivars. Fungal proteolytic and polygalacturanase activities were measured at different days postinoculation either with the pathogenic F. solani f. sp. eumartii, isolate 3122 or with the non‐pathogenic F. solani, isolate 1042. After inoculation with the pathogenic fungus, proteolytic and polygalaturonase activities were higher in the susceptible than in the resistant cultivar. In addition, we found a correlation between the levels of proteolytic activity detected in the intercellular washing fluids with the size of the lesion area caused by F. solani f. sp. eumartii in Huinkul tubers. The action of the proteolytic activity over cell wall proteins of both potato cultivars was assayed. An extracellular potato protein with homology to proteinase inhibitors of the Kunitz family was identified as a substrate of the proteolytic activity in the susceptible cultivar. A microscopic study revealed differences between the potato genotypes in the rate of response to infection by F. solani f. sp. eumartii. In addition, the cell wall alteration caused by F. solani f. sp. eumartii in cortical cells of susceptible tubers was evaluated. The data with respect to the correlation between the course of cyto‐ and biochemical events of the two host–pathogen interactions were discussed.     

甘薯根腐病菌侵染对甘薯内源激素水平的影响

甘薯根腐病病原菌[Fusarium solani(Mart.)Sacc.f.sp.batatas McClure,简称FSB]侵染及其培养液滤液处理高敏感性甘薯品种‘胜利百号’后,引起甘薯叶片、茎尖和根部组织内源ABA含量大幅升高。其中在根部出现最早,但茎尖中积累浓度最高。侵染后甘薯叶片、茎尖和根部组织内源GA1/3含量显著低于对照。甘薯组培苗经FSB培养滤液处理9h后,ABA含量显著上升,处理15h,ABA含量呈下降趋势,而GA1/3含量在101和102稀释液处理15h(103稀释液处理12h)时出现显著上升。这些结果有助于解释甘薯根腐病株矮小不产生藤蔓,并在秋季大量现蕾开花的生理现象。     

Potato Cultivar, Pathogen Isolate and Antagonist Cultivation Medium Influence the Efficacy and Ranking of Bacterial Antagonists of Fusarium Dry Rot

The process of selecting biological control agents for further development frequently does not involve conducting bioassays of strain effectiveness on a range of pathogen isolates or host cultivars. Additionally, though previous studies have demonstrated that the medium used to produce biomass of an antagonist can alter its efficacy, this factor is also rarely considered when selecting for the most effective antagonist. Host cultivar, pathogen isolate, and the cultivation medium used to produce the antagonists' biomass were examined as factors of potential importance for assessing the relative effectiveness of bacterial biocontrol strains accurately. Five bacterial antagonists that control Fusarium dry rot on stored potato tubers were assayed for effectiveness against 10 isolates of Gibberella pulicaris . All antagonists reduced disease severity (35-81%) regardless of the specific assays conducted. However, when the antagonists' biomass were produced on two media that differed both in nutrient composition and phase, the efficacy ranking of antagonist Enterobacter sp. S11:P:08 varied from first to fourth most effective. For the antagonists studied, the phase of a nutritionally identical medium had little impact on the efficacy ranking of the five antagonists. Four of the five antagonists had efficacy rankings that ranged from first to last depending on the isolate of the pathogen used to conduct the bioassay. The cultivar of the host also caused variations in the efficacy ranking of the antagonists. These results indicate that bioassays should be conducted using a range of liquid culture production media, pathogen isolates, and host cultivars in order to choose an antagonist that has the highest likelihood for commercial development as an effective biological control product.     

An improved test for evaluating the pathogenicity of isolates of Fusarium solani f.sp. pisi on pea

An improved in vitro test is described for determining the pathogenicity of Fusarium solani f.sp. pisi isolates on pea. This technique involves the use of polypropylene fibre Milcap plugs to suspend peas in boiling tubes containing spore suspensions in 0.1% water agar. Results were available after 14 days of incubation at 25°C. Four levels of pathogenicity were detected on pea cultivars Little Marvel and Dark Skinned Perfection using a total of eight isolates and strains of F. solani f.sp. pisi.     

Comparison of Screening Methods for Resistance to Fusarium Root Rot in Common Beans (Phaseolus vulgaris L.)

Root rot, caused by Fusarium solani f. sp. phaseoli, is one of the main root diseases impacting production of common beans throughout the world. Because resistance of common beans to root rot is a quantitative trait that is strongly influenced by environmental factors, reproducible methods to screen bean plants for resistance to root rot are critical to the selection process. In this study, we adapted the inoculum layer method (ILM) developed for screening soybeans for resistance to Phytophthora rot and compared it with the traditional liquid inoculum method (LIM) for screening common beans for resistance to Fusarium root rot. In addition, two methods of evaluating resistance using the ILM were compared. The most significant Pearson correlation coefficient between trials involving 80 recombinant inbred lines was achieved with the ILM and counting discoloured vascular bundles in the lower stem (r_p = 0.7113***) compared to rating the discoloration on root and hypocotyl (r_p = 0.5555***). The traditional (LIM) screening method and rating the discolouration on roots resulted in a non‐significant correlation between trials (r_p = 0.1084).     

Efficacy of combined applications of antagonist bacteria and chemical resistance inducers for the management of Fusarium solani causing root rot in Withania somnifera

Application of Thiosalicylic acid+Bacillus cereus; O-Acetylsalicylic acid+Pseudomonas fluorescens reduced root rot severity by 85 and 88% and enhanced root yields by 358 and 419%, respectively, against Fusarium solani induced root rot disease in Withania somnifera. Reduction in disease severity was correlated with defence-related enzymes peroxidase, polyphenol oxidase and phenyl ammonium lyase.     

Effect of chitosan for the control of potato diseases caused by Fusarium species

The antifungal activity of chitosan against Fusarium spp. was investigated based on in vitro and in vivo assays, and its possible modes of action were also explored. Chitosan applied at 4.0 g/L of acetic acid-distilled water solution significantly decreased the mycelial growth of Fusarium oxysporum, Fusarium sambucinum and Fusarium graminearum by 88.4%, 89.0% and 89.8%, respectively. Tuber treatment by chitosan (4.0 g/L) of acetic acid-distilled water solution, prior to inoculation, reduced dry rot severity induced by F. oxysporum and F. sambucinum by 60.0% and 48.2%, respectively. When tested as plant treatment, potato plants inoculated with Fusarium species, exhibited 33.5%–45.3% less wilting severity as compared to the control. This abiotic treatment improved the phenolic compounds activities and defence-related enzymes such as peroxidase and polyphenoloxidase in potato tubers inoculated with Fusarium spp. Results clearly demonstrated that chitosan could be explored as an alternative agent to chemical fungicides for the control of tuber dry rot and Fusarium wilt through induction of the plant defence system.     

Application of Soil‐borne Actinomycetes for Biological Control against Fusarium Wilt of Chickpea (Cicer arietinum) caused by Fusarium solani fsp pisi

Black root rot, caused by Fusarium solani f.sp. pisi, is a devastating soil‐borne disease in chickpea in Iran with no effective control measures. With the aim of finding applicable biocontrol agents to alleviate the malady, isolates of Actinomycetes isolated from soil and their antagonistic effect against F. solani f.sp. pisi were evaluated both in vitro and in vivo. More than 100 Actinomycetes isolates were screened for their antifungal activities against the pathogen. The most active isolates were evaluated in greenhouse for their biocontrol performance. Based on the results of dual cultures in screening evaluations, the size of inhibition zone of fungal growth, and the most effective antagonist isolates (S3, S12 and S40) were selected for further studies. Identity of active isolates was determined, in this regard, 16S rDNA of isolates were amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then performed using NCBI BLAST method. Comparison of the near full length 16S rRNA sequence of isolates to GenBank sequences demonstrated that isolates S3 and S12 were most similar to Streptomyces antibioticus, while isolate S40 was most similar to Streptomyces peruviensis. Biocontrol studies of these isolates in control of the disease in greenhouse significantly decreased the disease severity. Actinomycetes isolate S12 demonstrated the greatest effect in reducing disease than the other two. Results of this research are at preliminary stage for developing biocontrol agents. These data can be utilized as a platform for future studies with the aim of commercializing these biocontrol products and hoping to step towards sustainable agriculture.     

Induced Resistance in Cucumbers against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG2–2 by Infection of the Cotyledons

Localized infection in cucumber cotyledons with Colletotrichum lagenarium induced resistance against infection after challenge inoculation with Rhizoctonia solani AG2–2 and Fusarium oxysporum f. sp. cucumerinum in the roots. The plants were unprotected in soil that was infested heavily with R. solani or in contact with the mycelium, and induced resistance was not observed. Wounding of the root also negated the effect of induced resistance to F. oxysporum .     

Development of amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot

Aims

The objective of this work was to design an amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil.

Methods and Results

Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non‐PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils.

Conclusions

The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen.

Significance and Impact of the Study

These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas.     

An extracellular enzyme from Fusarium solani f. sp. phaseoli which catalyses hydration of the isoflavonoid phytoalexin, phaseollidin

Among the antimicrobial phytoalexins produced by Phaseolus vulgaris (French bean) are the prenylated isoflavonoids kievitone and phaseollidin. Two enzyme activities, kievitone hydratase and phaseollidin hydratase, occur in culture filtrates of the bean pathogen, Fusarium solani f. sp. phaseoli, and catalyse similar hydration reactions on the dimethylallyl moieties of the phytoalexins. The enzymes nearly co-purified during hydroxyapatite chromatography followed by preparative native gel electrophoresis. Eluates from successive slices taken from the native gel were assayed for both activities. Although they were not completely separated in the native gel, the activity profiles indicated that the two activities were distinct. The Km of phaseollidin hydratase for phaseollidin was approximately 7 microM.     

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5 r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 9 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in C 90%reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.     

Alternative Hosts for Fusarium spp. Causing Crown and Root Rot of Asparagus in Spain

Asparagus crown and root rot caused by Fusarium oxysporum f.sp. asparagi (Foa), F. proliferatum (Fp) and F. solani (Fs) result in early decline and loss of crop production. The role of several crop species on the survival of the Fusarium spp. was investigated. The root symptoms and plant weight of seven crop species were evaluated after inoculation with each of the three Fusarium spp. The number of colony‐forming units of the Fusarium spp. from root tissues was also determined. Garlic was shown to be a symptomatic host for Foa, Fp and Fs; Fs was also pathogenic to onion. Root colonization of garlic, onion, maize, wheat, potato and sunflower suggested that they are reservoirs of Foa, Fp and Fs from asparagus and demonstrated the importance of crop rotation on the development of this asparagus disease.     

Assessment of the bioregulatory activity of the leaf juices of higher plants in Al-Taif,Saudi Arabia against Fusarium solani,Phytophthora spp. and Rhizoctonia solani

Resistance to conventional fungicides causes poor disease control in agriculture. Natural products from plants have great potential as novel fungicide sources for controlling pathogenic fungi. In this study the antipathogenic activity of the leaf juices of 11 plant species (Chenopodium ambrosioides, Pulicaria vulgaris, Lavandula pubescens, Lavandula dentata, Ageratum conyzoides, Ficus retusa, Zizyphus nummularia, Acacia tortilis, Phragmanthera sp. Aff. Rufescens, Lawsonia alba and Olea europaea) were evaluated in vitro against three plant pathogenic fungi (Fusarium solani, Phytophthora spp. and Rhizoctonia solani). Plate assays showed that the leaf aqueous juices have antifungal activity against these fungi. The aqueous extracts of Pulicaria vulgaris, Lavandula dentata, Ageratum conyzoides, Ficus retusa, Zizyphus nummularia, Acacia tortilis, Phragmanthera sp. Aff. Rufescens (when associated with Acacia tortilis), Lawsonia alba and Olea europaea exhibited antifungal properties against Fusarium solani, Phytophthora spp. and Rhizoctonia solani with variable degrees. On the other hand, Chenopodium ambrosioides, Lavandula pubescens and Phragmanthera sp. Aff. Rufescens (when associated with Zizyphus nummularia) did not exhibit any fungitoxicity. All these observations suggest the possible exploitation of Chenopodium oil as a potential botanical fungitoxicant in ecofriendly control of post-harvest biodeterioration of food commodities from storage fungi.     

High-performance liquid chromatography methods for the analysis of adrenergic amines and flavanones in Citrus aurantium L. var. amara

Reverse-phase HPLC coupled with photodiode array detection was used for the simultaneous separation and determination of naturally occurring adrenergic amines (octopamine, synephrine and tyramine) in fruits and dry extracts of Citrus aurantium L. var. amara and in herbal medicines derived therefrom. Synephrine was the main component in fruits (0.10-0.35%) and in dry extracts (3.00-3.08%) and was present in the range 0.25-0.99% in herbal medicines. Flavanones were analysed in the same samples using a reverse-phase HPLC technique which allowed the identification and quantification of neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, naringenin and hesperetin. C. aurantium fruits and derivatives contained mainly glycosylated flavanones: in particular, naringin and neohesperidin were found to be the major flavonoids and their concentrations ranged from 1.80 to 26.30 and from 3.90 to 14.71 mg/g, respectively. The levels of aglycones were very low in all samples tested.     

Evaluation of a Peroxyacetic Acid Disinfectant in Hot-water Treatment for the Control of Basal Rot (Fusarium oxysporum f. sp. narcissi) and Stem Nematode (Ditylenchus dipsaci) in Narcissus

Formaldehyde is used routinely in the hot-water treatment (HWT) of narcissus bulbs for the control of stem nematode (Ditylenchus dipsaci) and basal rot (caused by Fusarium oxysporum f.sp. narcissi). Formaldehyde is unpleasant for operators to use, and does not kill all of the thick-walled chlamydospores of F. oxysporum and so less hazardous but more effective materials are being sought. Peroxyacetic acid (as Jet 5, a commercial disinfectant containing 5% peroxyacetic acid) was evaluated in vitro and in a field trial as a possible alternative to formaldehyde. In laboratory studies, peroxyacetic acid (as 1% Jet 5) was as effective as formaldehyde (as 0.5% commercial formalin containing 38 to 40% formaldehyde) in killing free-swimming stem nematodes and nematodes in the wool stage. Peroxyacetic acid (as 0.5% Jet 5) killed F. oxysporum chlamydospores within 1 h, whereas total kill was not achieved with formaldehyde (concentration as above) after 4 h. In a 2 year field trial, there was no evidence of detrimental effects on a healthy narcissus stock due to using peroxyacetic acid. In an infested, diseased stock, bulbs were virtually destroyed by stem nematode within 2 years when HWT was not given. The greatest reduction in nematode symptoms, and the highest bulb yields, were found when formaldehyde or the higher rates of peroxyacetic acid were used in combination with thiabendazole.     

Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.