Vegetative compatibility groups of Japanese isolates ofVerticillium dahliae

Japanese isolates ofVerticillium dahliae were examined for vegetative compatibility relationships using nitrate-nonutilizing mutants. Four levels of vegetative compatibility were differentiated according to the degree of compatibility between the tester mutants ofnit1 and NitM. Wild-type growth with a complementation line greater than 5 mm wide was defined as “strong reaction (++)”, i.e., compatible. Ten out of 15 isolates showed compatibility and were separated into three groups, provisionally designated as VCGJ1, VCGJ2, and VCGJ3, depending upon their reactions. This method was used to estimate, genetic diversity within a local population ofV. dahliae. Another 12 isolates from Gunma Pref. were paired with tester isolates of the three vegetative compatibility groups proposed. Eight Gunma isolates were assigned to VCGJ1 or VCGJ2. Two isolates were incompatible with all testers. The remaining 2 isolates were self-incompatible. Thus, 18 out of 27 Japanese isolates ofV. dahliae were assigned to VCGs: 8 to VCGJ1, 7 to VCGJ2, and 3 to VCGJ3. VCGJ1 was compatible with both VCGJ2 and VCGJ3, but VCGJ2 and VCGJ3 showed a weak reaction with each other. Japanese isolates ofV. dahliae were thus demonstrated to form a VC group comprising three subgroups.     

Relationship of inoculum ofVerticillium dahliae to disease in tomato plants

Summary Tomato (Lycopersicon esculentum Mill.) seedlings were inoculated by dipping roots in suspensions of conidia ofVerticillium dahliae Kleb. There was a linear relationship between inoculum concentration and infection at 21 days after inoculation in steam-disinfested soil. Thereafter the number of infections per plant increased. At higher concentrations data indicated a synergistic interaction between conidia. Synergism was more pronounced and was detected earlier in soil not disinfested before inoculation. When conidia from 3-, 7-, and 14-day-old cultures were used, the greatest response was from 3-day-old cultures. A lower total response and lower rate of response to older inoculum indicated a decrease in aggressiveness of conidia with age. Older plants were less affected by the pathogen when plants inoculated at the fourth and sixth leaf stages with minimum root disturbances were compared. There was also an increase in infection with an increase in the volume of root zone infested. In field experiments infection reduced stand when roots were disturbed at inoculation. With minimum root disturbance yields were reduced without a reduction in stand. Part of thesis to be submitted by the senior author to the University of Stellenbosch in partial fulfilment of the Ph.D. (Agric.) degree.     

Assessment of vegetative compatibility of race-2 tomato wilt isolates ofVerticillium dahliae in Japan

Verticillium dahliae race-2 can invade the resistant cultivars of tomato possessing theVe gene. This new race was recently found in several regions in Japan, and 10 isolates ofV. dahliae race-2 from these regions were used in our study. Pathogenicity tests identified these isolates as the tomato pathotype (B). We examined the vegetative compatibility of 8 of these 10 Japanese isolates ofV. dahliae race-2 to estimate their genetic relatedness with the testers of Japanese vegetative compatibility group previously proposed (VCGJ) usingnit mutants. Compatiblenit1 and NitM mutants were obtained from allV. dahliae race-2 isolates. Selected representativenit1 and NitM mutants of eachV. dahliae race-2 isolates were paired with VCGJ testers. All isolates ofV. dahliae race-2 showed a strong reaction with VCGJ2, i.e., tomato pathotype. All isolates ofV. dahliae race-2 except for isolate To22 reacted weakly to VCGJ1 and J3. Japanese isolates ofV. dahliae race-2 were assigned as VCGJ2 and were hence vegetatively closely related with those ofV. dahliae race-1. The origin of Japanese isolates ofV. dahliae race-2 was discussed.     

Germination and production ofMacrophomina phaseolina microsclerotia as affected by oxygen and carbon dioxide concentration

Summary Germination of microsclerotia ofMacrophomina phaseolina was observed at O₂ concentrations of 16% or higher in autoclaved soil. Germination was delayed but otherwise unaffected as O₂ decreased from 21 to 16% and was in all cases complete in 32 hours. Laboratory-produced microsclerotia consistently germinated more rapidly and seemed more independent of O₂ concentrations within the range that permitted germination than naturallyproduced microsclerotia.Population changes in soil as measured by microsclerotial counts were inversely correlated with depth of interment and reduced O₂ concentration. Our inability to detect significantly growth responses ofM. phaseolina in non autoclaved soil was apparently related to limited O₂ although other possibilities are discussed.Contribution of the Missouri Agricultural Experiment Station Scientific Journal Series No. 9124.     

Specific binding of a Verticillium dahliae phytotoxin to protoplasts of cotton, Gossypium hirsutum

Summary The occurrence of specific, high-affinity binding sites for a protein-lipopolysaccharide (PLP) phytotoxin purified from culture filtrates of a virulent Vertidllium dahliae isolate has been demonstrated in cotton protoplasts. Binding of the ¹²⁵I-radiolabelled PLP-complex to protoplasts from cotyledon tissue was saturable and with an affinity (K_d = 17.3 nM) comparable with the concentration required for biological activity. A single class of binding site, accessible at the surface of the intact protoplasts, was found and the maximal number of binding sites were estimated as 2.41 × 10^–16 moles per protoplast. The binding affinity to protoplasts proved near identical to that found with purified plasma membrane fractions from roots. When cultivars exhibiting resistance or susceptibility towards the pathogen were compared, no significant differences were found in the affinity of binding, but five times as many binding sites per protoplast and sixteen times as many binding sites per mg membrane protein were found in the resistant cultivar.Abbreviations PLP protein-lipopolysaccharide - k_d dissociation constant - B_max maximal number of binding sites - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Karyotypic variability in plants of Solanum melongena regenerated from callus grown in presence of culture filtrate of Verticillium dahliae

Summary Plantlets were regenerated from calli derived from leaf expiants of three genotypes of Solanum melongena (two parental genotypes and their hybrid). The cytological analysis showed that a) plants regenerated were all mixoploid, b) toxic medium (basal medium added with filtrate culture of Verticillium dahliae) was able to evidence karyotypic differences between genotypes not displayed by plants regenerated from callus grown on control medium, c) chromosomal mosaicism persists up to plant maturity and also in the selfed progeny. The results are discussed in terms of a selective process involving genes controlling chromosome number and/or a direct effect of toxic medium on the activity of the same genes.This research is supported by a grant from ERSO (Ente per la Ricerca e Sperimentazione in Ortoflorifrutticoltura e Sementi) — Regione Emilia Romagna     

Verticillium dahliae toxin induced alterations of cytoskeletons and nucleoli in Arabidopsis thaliana suspension cells

Summary. In plant cells, cytoskeletons play important roles in response to biotic and abiotic stresses. However, little is known about the dynamics of cytoskeletons when cells are attacked by unphysical stress factors such as elicitors and toxins. We report here that the toxin of Verticillium dahliae (VD toxin) induced changes of microfilaments (MFs) and microtubules (MTs) in Arabidopsis thaliana suspension-cultured cells. When cells were treated with a low concentration of VD toxin, MFs were disrupted ordinally from the cortex to the perinuclear region, and then recovered spontaneously; but the MTs persisted. The MFs in the perinuclear region showed more resistance to VD toxin than the cortical ones. In contrast, when cells were treated with a high concentration of VD toxin, MFs and MTs were disrupted sooner and more severely and did not recover spontaneously. Treatments with high concentrations of VD toxin also induced changes of nucleoli. At the early stages of treatment, a nucleus had a single ring-shaped nucleolus. At the later stages, multiple smaller and more brightly fluorescing nucleoli emerged in a single nucleus. Disrupted MFs could be recovered by removing the VD toxin before the ringshaped nucleoli appeared. All these results showed that MFs and MTs play important roles in the early defense responses against VD toxin in Arabidopsis suspension cells. The cytoskeletons may be used as sensors and effectors monitoring the defense reactions. The changes of nucleoli induced by VD toxin should be important characteristics of cell death. Correspondence and reprints: Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing 100094, People’s Republic of China.     

Moderate drought influences the effect of arbuscular mycorrhizal fungi as biocontrol agents against Verticillium-induced wilt in pepper

Previous studies have shown that the arbuscular mycorrhizal fungus (AMF) Glomus deserticola (Trappe, Bloss and Menge) can diminish the negative effect of Verticillium dahliae Kleb. on pepper yield. On the other hand, it is known that AMF can be more beneficial for plant growth and physiology under dry conditions than when soil moisture is plentiful. Therefore, our objective was to assess if a moderate water deficit imposed on pepper plants before their inoculation with V. dahliae could improve the effectiveness of G. deserticola as biocontrol agent. In the present experiment, the delay in disease development in Verticillium-inoculated plants associated with AMF did not occur under well watered conditions. In addition, the establishment of mycorrhizal symbiosis and the development of structures by AMF were delayed when both symbiotic and pathogenic fungi infected the same root. Therefore, it is suggested that the equilibrium between pepper plant, G. deserticola and V. dahliae is so complex that small changes in competition between symbiotic and pathogenic fungi for host resources can modify the efficiency of AMF as a biocontrol agent. On the other hand, water deficit enhanced the deleterious effect of V. dahliae on fruit set and yield only when pepper plants were not associated with G. deserticola, which reinforces the idea that AMF may be more important for host plants subjected to stressful conditions. However, comparing well watered non-mycorrhizal and predroughted mycorrhizal plants, we found that moderate water deficit imposed before inoculation with V. dahliae did not improve the effectiveness of G. deserticola as a biocontrol agent.     

Ultrastructure and germinability of Verticillium dahliae microsclerotia parasitized by Talaromyces flavus on agar medium and in treated soil

The interactions between microsclerotia (ms) of the fungal plant pathogen Verticillium dahliae and the mycoparasite Talaromyces flavus were followed in soil and on agar medium. Germinability of ms, which had been incubated for 14 days in soil treated with 0.5% of a T. flavus ‐ wheat bran preparation, decreased from 84% to 17%, as compared with 81% and 74% in untreated soil and in soil treated with a sterilized biocontrol preparation respectively. Germinability of ms which had been buried in treated soil for 4 days decreased to 70%, all ms being parasitized by T. flavus. Upon transfer of the ms to untreated soil for 10 more days, germinability decreased further to 20%, indicating that T. flavus continued to parasitize sclerotia in the untreated soil. Scanning electron micrographs showed heavy fungal colonization and typical T. flavus conidia on the surface of the ms buried in the treated soil, but not in control soils. Transmission electron micrographs of ms incubated with T. flavus on agar revealed parsitism involving invasion of some host cells by means of small penetration pegs; the host cell walls were mainly lysed at their site of contact with the parasite hyphal tips. Further colonization of the ms cells occurred simultaneously with the degradation of the invaded host cell contents, rather than the cell walls. Mycoparasitism of V. dahliae ms by T. flavus hyphae may be involved in the biological control of verticillium wilt disease.     

Defence-related Enzymes in Pepper Roots During Interactions with Arbuscular Mycorrhizal Fungi and/or Verticillium dahliae

Previous studies have described that arbuscular mycorrhizal fungi (AMF) can reduce the deleterious effect of Verticillium dahliae Kleb. on pepper growth and yield. In mycorrhizal plants, the bioprotection against soil-borne pathogens can result from the preactivation of defence responses that include some structural modifications and the accumulation of Pathogenesis-Related (PR) proteins. Our first objective was to study if V. dahliae induced defence mechanisms in roots before infected pepper developed visible symptoms of disease. The second aim was to determine if AMF induced defence-related enzymatic activities in pepper roots before or after pathogen’s attack. Results showed that the colonization of pepper roots by Glomus deserticola (Trappe, Bloss and Menge) induced the appearance of new isoforms of acidic chitinases, superoxide dismutase (SOD) and, at early stages, peroxidases. In contrast, V. dahliae neither stimulated the phenylpropanoid pathway nor elicited hydrolytic activities in infected pepper roots. Only in mycorrhizal plants, the inoculation with V. dahliae slightly increased both phenylalanine ammonia-lyase (PAL) and peroxidase activities two weeks later. Mycorrhizal-specific induction of new isoforms of acidic chitinases and SOD together with enhanced peroxidase and PAL activities 2 weeks after pathogen inoculation could be involved in the biocontrol of Verticillium-induced wilt in pepper by AMF.     

Seedling vaccination by stem injecting a conidial suspension of F2, a non-pathogenic Fusarium oxysporum strain, suppresses Verticillium wilt of eggplant

Several bacterial and fungal strains have been evaluated as biocontrol agents (BCAs) against Verticillium dahliae. In these studies, the BCAs were applied as a root drenching inoculum; however, this application method may have an adverse effect on the native, beneficial for the plants, microbial community. In the present study, it was evaluated whether endophytic application by stem injecting a conidial suspension of the non pathogenic Fusarium oxysporum F2 strain, isolated from a V. dahliae suppressive compost amendment, could reduce significantly Verticillium wilt symptom development in eggplants. It was revealed that stem injection of F2 seven days before transplanting the seedlings to soil infested by V. dahliae microsclerotia resulted in reduced disease severity compared to the control treatment. To visualise F2 ramification into the plant vascular system eggplant stems were injected with an EGFP transformed F2 strain. It was shown that F2 colonises effectively the plant vascular tissues over a long period of time as it was assessed by F2 DNA levels. In parallel, qPCR analysis showed that the application of F2 reduced significantly the amount of V. dahliae DNA in the stem tissues compared to the control treatment.     

Effect of vesicular-arbuscular mycorrhizal fungi on verticillium wilt of cotton

The development of vesicular-arbuscular mycorrhizal fungi (VAMF): Glomus mosseae (Nicol and Gerd.) Gerdemann and Trappe, Glomus versiforme (Karsten) Berch, Sclerocystis sinuosa Gerdemann and Bakhi and Verticillium dahliae and the effects of the VAMF on the verticillium wilt of cotton (Gossypium hirsutum L. and Gossypium barbadense L.) were studied with paper pots, black plastic tubes and clay pots under natural growth conditions. All of the tested VAMF were able to infect all the cotton varieties used in the present experiment and typical vesicles and arbuscules were formed in the cortical cells of the cotton roots after inoculation. The cap cells, meristem, differentiating and elongating zones of the root tip were found to be colonized by the VAMF. In the case of most V. dahliae infection, the colonization occurred mostly from the root tip up to 2 cm. VAMF and V. dahliae mutually reduced their percentage of infection when inoculated simultaneously. VAMF inoculation reduced the numbers of germinable microsclerotia in the soil of the mycorrhizosphere, while the quantity of VAM fungal spores in the soil was not influenced by infection of with V. dahliae. The % of arbuscule colonization in roots was negatively correlated with the disease grades, while the numbers of vesicles in roots were not. These results suggest that certain vital competition and antagonistic reactions exist between VAMF and V. dahliae. VAMF reduced the incidence and disease indices of verticillium wilt of cotton during the whole growth phase. It is evident that cotton seedling growth was promoted, flowering was advanced, the numbers of flowers and bolls were increased, and this resulted in an increase in the yield of seed cotton. Among the VAMF species, Glomus versiforme was the most effective, and Sclerocystis sinuosa was inferior. So far as the author is aware, such an effect of VAMF on the increase of cotton wilt tolerance/resistance is reported here far the first time.     

Biocontrol of tomato wilt by Penicillium oxalicum formulations in different crop conditions

Eight formulations of Penicillium oxalicum (FOR1 to FOR8) were obtained by the addition of various ingredients, in two separate steps of the production and drying of P. oxalicum conidia. These formulations were then evaluated against tomato wilt in three glasshouse (G1 to G3) and two field (F1 and F2) experiments. All formulations were applied to seedlings in seedbeds 7 days before transplanting at a rate of 10⁷ spores g⁻¹ seedbed substrate. The conidial viability of each formulation was estimated by measuring germination just after fluid bed-drying, before seedbed application and after 1 and 2 years of storage at 4 °C under vacuum. The densities of P. oxalicum were estimated in the seedbed substrate and in the rhizosphere of three plants per treatment just before transplanting. Initial conidial viability of formulations just after fluid bed-drying was approx. 80%, except for FOR1, FOR4, and FOR7 which were 60%. The initial viability was maintained up to 40–50% for 2 years of storage at 4 °C under vacuum, except for FOR1. All formulations had 50% viability at application time. Populations of P. oxalicum in the seedbed substrate just before transplanting were >10⁶ cfu g⁻¹ soil in G3 and F2; populations in rhizosphere were also >10⁶ cfu g⁻¹ fresh root, except for FOR3, FOR5, and FOR6 in G2. A range of 22–64% of disease reduction was observed with all formulations, although these reductions were not significant (p = 0.05) for FOR1, FOR4, and FOR5 in any experiment. Contrast analysis showed significant differences between biological treatments and untreated control (p = 0.05) in all experiments, but no significant differences between biological and chemical treatments. Initial conidial viability of P. oxalicum in formulations and populations of P. oxalicum in the seedbed substrate explained 78.26% of the variability in P. oxalicum populations in tomato rhizosphere before transplanting. Disease incidence in untreated plants was negatively correlated (r = −0.54) with the percentage of disease control. The relationship between the viability of formulations, the populations of P. oxalicum in seedbed and rhizosphere, and the control of tomato wilt is discussed.     

Application ofVerticillium lecanii for the control ofAphis gossypii by a low-volume electrostatic rotary atomiser and a high-volume hydraulic sprayer

Verticillium lecanii (Fungi: Deuteromycete) blastospores were applied to a chrysanthemum crop by an ULV electrostatically charged rotary atomiser (APE-80). The deposition of spores and subsequent control ofAphis gossypii were compared to high volume hydraulic application. A full rate treatment (2×10¹³ blastospores per ha.) was applied by the APE-80 at week 1 and reduced spore rates of 1/6^th and 1/12^th applied by both the APE-80 and the hydraulic sprayer once and twice a week respectively for weeks 1 to 6. Untreated plots served as controls. Initial deposits of spores were higher with the electrostatic sprayer and better distributed with respect to the position of the target aphids. Significantly lower aphid populations were recorded on the electrostatically treated plots in week 4. The single full rate treatment had significantly fewer aphids than the untreated plots from week 3 and all treatments had significantly fewer aphids than the untreated plots from week 5 onwards. The proportion of the aphid population killed byV. lecanii was higher on the electrostatically treated plots until week 6.      

Interactions between iron nutrition and Verticillium wilt resistance in tomato

The relationship between Fe nutritional status and Verticillium wilt disease in tomatoes possessing single gene resistance to Race 1 of Verticillium dahliae was investigated using hydroponic culture media. Iron limiting conditions increased the sensitivity of resistant tomatoes to the pathogen as expressed by wilting and chlorosis. Distance of fungal vascular invasion was approximately the same in both Fe replete and Fe limited treatments. Comparison of near-isolines revealed that the magnitude of disease expressed in Fe deficient Pixie II (resistant) was considerably less than that expressed by the susceptible Pixie variety. Infection of tomato did not enhance the severity of low-Fe stress as quantified by root peroxidase activity and chlorophyll content of young leaves.     

Effects of soil acidity on nodulation ofAlnus glutinosa and viability ofFrankia

Summary Black alder seedlings were grown from seed for 7 weeks in six soils limed to various pH levels and inoculated withFrankia in two inoculation-seeding time combinations (inoculated and seeded concurrently; inoculated then seeded 5 weeks after inoculation). Three mine soils and three non-mine soils were used. Soil pHs in the study ranged from 3.6 to 7.6. In the second inoculation-seeding time combination, a series of soil samples at each of the pH levels below 7.0 were relimed to pH 7.0 immediately prior to seeding. The purpose of the study was to examine the effects of soil acidity on the nodulation of black alder byFrankia and the viability ofFrankia in acid soils. Based on the average number of nodules established per seedling, soil pH was determined to be a significant factor affecting nodulation in the mine soils. The highest levels of nodulation occurred between soil pH 5.5 and 7.2. Below pH 5.5, nodulation was reduced. There was also evidence of decreased viability of the endophyte below pH 4.5.     

Quantitative assessment of the interaction between the antagonistic fungus Talaromyces flavus and the wilt pathogen Verticillium dahliae on eggplant roots

Quantitative aspects of the interaction between the antagonist Talaromyces flavus, the pathogen Verticillium dahliae and eggplant roots, were studied. When eggplant roots were inoculated with T. flavus, prior to the infection with the pathogen, the population density of T. flavus on V. dahliae-infected roots was at least 3 times higher than on healthy uninfected roots, and the proliferation of T. flavus on diseased eggplant roots was related to the severity of wilt symptoms, in the two levels of application of T. flavus studied. However, in all classes of disease severity tested (disease index, 0–3), the population density of T. Flavus on eggplant roots treated with 10⁶ ascospores g^–1 rooting mixture was significantly (p=0.05) higher than with 10⁵ ascospores g^–1. In roots treated with 10⁵ and 10⁶T. flavus ascospores g^–1 rooting mixture, the population density of V. dahliae was reduced by 51% and 69%, respectively. When testing the relationships between the population density of V. dahliae in the roots and disease severity, no significant (p=0.05) difference was found between disease indexes 2 and 3. However, the density of V. dahliae on roots of plants with disease index 1 was significantly (p=0.05) lower than disease indexes 2 and 3. The positive relationship between the inoculum concentration of V. dahliae and the population density of T. flavus developed on eggplant roots was significant (p=0.001), linear, and highly correlated (r=0.945) on a logarithmic scale. In addition, the analysis of these data revealed a significant (p=0.05), high, negative and linear correlation (r=–0.985) between the log concentration of V. dahliae inoculum and the disease reduction achieved by T. flavus.     

Heat treatment and viability assessment by Evans blue in cultured Symbiodinium kawagutii cells

Evans blue was used to determine viability in Symbiodinium kawagutii cultures but heat treatments at up to 80°C for 1 h were required for detecting blue-stained dead cells. About 3 h at this temperature was necessary to obtain ~50% blue-stained dead cells, and more than 6 h for 100%. In comparison, Trypan blue showed unusual elevated numbers of viable cells at times when more than 50% cells were dead by Evans blue parameters. Respiration decreased to 17 and 13.2% after 1 and 2 h at 80°C, respectively, and a marginal but still statistically significant 7.5% was scored after 3 h. Thus, cultured S. kawagutii cells were more resistant to heat-induced death than expected, and Evans blue was reliable for assessment of their viability.     

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro

The protozoan parasites Giardia duodenalis and Cryptosporidium parvum are common causes of diarrhoea, worldwide. Effective drug treatment is available for G. duodenalis, but with anecdotal evidence of resistance or reduced compliance. There is no effective specific chemotherapeutic intervention for Cryptosporidium. Recently, there has been renewed interest in the antimicrobial properties of berries and their phenolic compounds but little work has been done on their antiparasitic actions. The effect of various preparations of blueberry (Vaccinium myrtillus) extract on G. duodenalis trophozoites and C. parvum oocysts were investigated. Pressed blueberry extract, a polyphenolic-rich blueberry extract, and a commercially produced blueberry drink (Bouvrage) all demonstrated antigiardial activity. The polyphenol-rich blueberry extract reduced trophozoite viability in a dose dependent manner. At 167 μg ml⁻¹, this extract performed as well as all dilutions of pressed blueberry extract and the Bouvrage beverage (9.6 ± 2.8% live trophozoites remaining after 24 h incubation). The lowest dilution of blueberry extract tested (12.5% v/v) contained >167 μg ml⁻¹ of polyphenolic compounds suggesting that polyphenols are responsible for the reduced survival of G. duodenalis trophozoites. The pressed blueberry extract, Bouvrage beverage and the polyphenolic-rich blueberry extract increased the spontaneous excystation of C. parvum oocysts at 37 °C, compared to controls, but only at a dilution of 50% Bouvrage beverage, equivalent to 213 μg ml⁻¹ gallic acid equivalents in the polyphenolic-rich blueberry extract. Above this level, spontaneous excystation is decreased. We conclude that water soluble extracts of blueberries can kill G. duodenalis trophozoites and modify the morphology of G. duodenalis and C. parvum.