Survival of Sclerotia of Rhizoctonia solani AG3PT and Effect of Soil‐Borne Inoculum Density on Disease Development on Potato

Sclerotia produced by a single isolate of Rhizoctonia solani AG3PT were buried in small plot experiments to investigate the effects of sclerotial production method, soil type and burial depth on sclerotial viability in field soil. The factor with the greatest effect on sclerotial viability, defined as the percentage of sclerotia germinating on agar following retrieval, in all experiments was the duration of burial. After 18 months, on average across all experiments, 20% of retrieved sclerotia were viable. A comparison between sclerotia produced in vitro on malt yeast extract agar and in vivo using micropropagated tubers in field soil found no significant differences between the two production methods on sclerotial viability. Burial in field soil at 20‐cm depth was found to significantly reduce sclerotial viability to 50% compared to 60% at 5 cm. In two pot experiments, amending the growing medium and soil with increasing inoculum densities of R. solani was found to increase stem number, stem canker and black scurf severity regardless of whether this soil‐borne inoculum was derived from mycelium or sclerotia. Black scurf incidence and severity were assessed 30–32 days posthaulm destruction and found to be similar for a range of sclerotial soil‐borne inoculum densities (1.0 × 10^?1 g/kg d.w. soil to 6 × 10^?3 g/kg d.w. soil). The significance of these findings in relation to pathogen survival, detection in soil and disease development is discussed.     

Peanut-pod mycoflora and kernel infection

Summary The mycoflora in soil clinging to dry pods of peanuts of the Spanish variety Argentine was sampled in 2 experiments by serially washing pods for increasing periods in changes of sterile water. Of the 9 principal fungi found,Aspergillus niger, A. flavus, A. terreus, Rhizopus spp. andSclerotium bataticola were present initially in relatively small numbers and decreased rapidly in subsequent dilutions. This decrease paralleled a decrease in weight of suspended material and in percentage of soil and organic particles greater than 0.016 mm in size.Penicillium funiculosum, P. rubrum, P. citrinum, andFusarium spp. were found in large numbers and increased or slowly decreased in numbers in subsequent dilutions. In some instances variations in numbers followed trends of percentages of soil and organic particles less than 0.016 mm in size.When dry pods with this known mycoflora were allowed to hydrate over a 6-day period at 26°, 32°, or 38°C, there was extensive pod penetration and kernel infection byA. niger, A. flavus, S. bataticola andRhizopus spp. but not by other fungi. The degree ofA. flavus andA. niger infection increased with increasing temperatures.Approved by the Director as Journal Series Paper No. 135.     

Effect of Moisture and Temperature on the Survival of Sclerotia of Sclerotium rolfsii in Soil

The sclerotia of Sclerotium rolfsii Sacc. survived in natural soil for 225 days under controlled moisture at 50% water holding capacity (WHC) after which there was a progressive reduction in the population of viable sclerotia. At 390 days only 48% were recovered. Sclerotia survived well at moisture contents upto 75% WHC but at 100% the population declined rapidly and none were recovered after 60 days. The contents of the sclerotia were found to lyse without germination leaving hollow rinds. Such lysis was found to be favoured between 25 and 40°C. At and below 20°C no such lysis was recovered and more than 80% sclerotia were recovered even after 60 days.     

Comparison of the effect of antibiotic substances on sclerotia of Mycosphaerella ligulicola and Colletotrichum coccodes

Summary Sclerotia ofColletotrichum coccodes tolerated much higher concentrations of actidione in agar than did sclerotia ofMycosphaerella ligulicola. With increase in concentration of the antibiotic sclerotia of both species took longer to germinate. Increased resistance of both species to actidione developed after growth of a single generation on media containing the antibiotic. Sclerotia ofC. coccodes survived 5 days immersion in a bacterial culture filtrate whereas scleroia ofM. ligulicola ceased to be viable after a similar period.Sclerotia ofC. coccodes andM. ligulicola exhibited strand and loose types of formation respectively. The degree of resistance of these sclerotia to antibiotic substances was correlated with both longevity in soil and type of formation, but, in general, there is unlikely to be a relationship between structure of the sclerotium and longevity.     

Hydrocarbons,free fatty acids,and amino acids of sclerotia of Sclerotinia sclerotiorum

Summary Sclerotia of Sclerotinia sclerotiorum (Lib.) D By. were obtained from commercial pea-and bean-cleaning operations or grown on potato-dextrose agar and synthetic glucose-and sucrose-salts agar media. The crude fat (ether extract) content of sclerotia varied from 0.8 to 1.5%. Extraction and fractionation of the lipids followed by gas chromatographic analysis showed that sclerotia from pea cleanings contained one predominant hydrocarbon which was absent from sclerotia produced in the laboratory. Sclerotia from natural sources and grown in the laboratory contained a similar distribution of C₁₈ unsaturated free fatty acids, however, quantitative differences were noted. Palmitic, oleic and linoleic were the major free fatty acids of the laboratory-grown sclerotia while a high proportion of linoleic acid was also found in sclerotia from natural sources. Sclerotia were fractionated into water-soluble and water-insoluble fractions. After acid hydrolysis of the waterinsoluble fraction, both fractions were analyzed for amino acids. Twenty-one compounds, including 2 unknowns, were detected in the soluble fraction. The hydrolyzates contained 19 amino acids, including the same 2 unknowns. Two compounds tentatively identified as ornithine and -aminobutyric acid were found only in the water-soluble fraction. The relative amino acid composition of the water-insoluble fraction of sclerotia from various sources was fairly constant but the arginine content decreased on the synthetic media.     

Isolation and Pathogenicity of Rhizosphere Fungi of Cocoyam in Relation to Cocoyam Root Rot Disease

Cocoyam is the second most important staple crop of Cameroon and root rot is a destructive disease of this plant. Pythium myriotylum (Pm), Fusarium solani (Fs), and Rhizoctonia solani (Rs) were isolated from the rhizosphere of root rot affected cocoyams and from the soil of a cocoyam experimental field plot temporarily devoid of same in Mamu, Cameroon. Pm was isolated from the above soil by the cocoyam leaf disc baits. Fs and Rs were also isolated from the same soils by the water dilution method and from the roots of diseased cocoyams but were always associated with mycelial growth of Pm. Pathogenicity of Pm and in combinations with Fs or Rs or Fs + Rs all developed cocoyam root rot disease (CRRD) symptoms on 3– and 7–month old cocoyam plantlets 2–7 days after inoculation. Symptoms included rotted roots and wilting with general chlorosis of inoculated plantlets. No symptoms of CRRD were noted on cocoyam plantlets inoculated with Fs, Rs, Fs + Rs, and distilled water. Results indicated that CRRD is not caused by several pathogens but only by Pm. Pm isolates from the soils and roots of diseased cocoyams and those maintained in the ROTREP laboratory have significantly bigger diameter of mycelial colony growth in 24 h–period at 31 °C on lima bean sucrose agar, V–8 juice sucrose agar, and potato sucrose agar than on potato dextrose agar and 2 % water agar. The cocoyam plantlets were raised axenically from tissue culture of explants in the laboratory.     

Factors affecting the survival of sclerotia of Sclerotium cepivorum in the Fraser Valley of British Columbia

Sclerotia of Sclerotium cepivorum buried in muck soil in the Fraser Valley decayed with time. The rate of decay of sclerotia was influenced by local environmental conditions. A mixture of soil with sclerotia increased their survival but there was no difference in the rates of decay in three different soils. The decay was greatest during winter when Fraser Valley fields are often flooded. Sclerotial decay was also affected by pretreatment of the sclerotia. Dried sclerotia decayed significantly (P < 0.05) faster than sclerotia which had not been dried, a phenomenon which is apparently due to changes in micro-organisms on the sclerotia. Dried sclerotia which had been incubated in moist soil had fewer bacteria and more fungi than sclerotia which had been incubated in soil without being dried. The increase in fungi on the dried sclerotia was due to a dramatic increase in Trichoderma spp.     

Survival of rhizobia on arrowleaf clover seeds under stresses of seed-coat toxins,heat and desiccation

Eighteen strains of Rhizobium leguminosarum bv. trifolii that had been found to be heat- and desiccation-stress tolerant in soil were tested for their resistance to Trifolium vesiculosum, Savi (arrowleaf clover) coat toxins using an agar plate technique and for survival on seed. They were further tested for their tolerance to seed coat toxins in combination with heat and desiccation stresses. The zone of inhibition in agar ranged from no inhibition to 21 mm diameter of growth inhibition around seed. Inoculation on seed under conditions of 28 °C and 100% relative humidity for 7 days resulted in a range of survival from nearly 100% to less than 1%. The correlation between zone of growth inhibition and survival on seed was not statistically significant. Strains that were desiccation or heat tolerant in soil were not necessarily desiccation or heat tolerant on seed but strains that were heat tolerant in soil were better able to survive on glass beads at 37 °C than strains that were not heat tolerant in soil. The zone of rhizobial inhibition on an agar medium was not a valid method to screen for isolates better able to survive on seed and the ability of rhizobia to withstand heat or drought stress on seed was not related to their ability to survive in soil. This revised version was published online in August 2006 with corrections to the Cover Date.     

Carpogenic Germination of Sclerotia of Sclerotinia sclerotiorum after Periods of Conditioning in Soil

Periods of conditioning in soil reduced the length of the resting period needed before sclerotia of Sclerotinia sclerotiorum could germinate to form apothecia. Curves for germination of sclerotia were fitted by a form of the log-logistic equation and from this equation the time taken for 50% germination (x₅₀) was calculated. These x₅₀ values were used as the basis for comparing germinability of sclerotia collected from infected sunflower plants and others conditioned in soil, or moist vermiculite for various times. Sclerotia from sunflower roots germinated sooner than those from the stem cavities. Germinability increased with the length of the conditioning period. Conditioning in soil was more effective than in moist vermiculite.     

Isolation of Pythium Acanthicum,P. oligandrum,and P. periplocum from Soil and Evaluation of Their Mycoparasitic Activity and Biocontrol Efficacy Against Selected Phytopathogenic Pythium Species

Mycoparasitic Pythium species with spiny oogonia were surveyed in 50 Palestinian agricultural fields subject to different cropping practices using the Sclerotia Bait Technique (SBT) and the Surface-Soil-Dilution-Plate method (SSDP) with the selective VP3 medium. The mycoparasitic Pythium species were obtained from 21 (42%) soils using the SSDP method and from 37 (74%) soils using SBT. Pythium acanthicum and P. oligandrum were isolated by both methods, whereas P. periplocum was isolated only by the SBT. Using a newly modified dual plate culture method (MDPCM), the three mycoparasites showed varying antagonistic performance against several Pythium host species under a range of in vitro conditions. However, P. periplocum and P. oligandrum were found to be active biocontrol agents against P. ultimum, the damping-off organism of cucumber. This pathogen was antagonized, on thin films of water agar, by the three mycoparasites, and was moderately susceptible to P. periplocum while slightly susceptible to P. acanthicum and P. oligandrum. In direct application method in which antagonistic mycoparasites were incorporated into peat/sand mixture artificially infested with P. ultimum under growthroom conditions, Pythium oligandrum and P. periplocum (at 500 CFUg⁻¹) significantly improved seedling emergence and protected seedlings from damping-off. In the seed coating method, biocontrol by two types of seed dressing (homogenate- or oospore coated seeds), was comparable to that achieved by direct application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Introduction and recovery of Clavibacter michiganensis subsp. michiganensis from agricultural soil

Twelve phytopathogenic Clavibacter michiganensis subsp. michiganensis strains were introduced into non-sterile agricultural loam soil at an inoculum density of about log. 6.0 cfu g^–1 dry weight soil. The soil samples were incubated at 22°C under a 12h light, 12h dark cycle and the population densities followed over a 30-day period by plating subsamples of serial dilutions of soil on Brain Heart Infusion agar amended with 0.5% (w/v) yeast extract and 30 g mL^–1 nalidixic acid. In 5 soil samples C. michiganensis cfu were not detected after 30 days incubation. Initially, C. michiganensis cfu accounted for about 90% of the cfu recovered but decreased to less than 10% after 30 days. These results suggested that some C. michiganensis strains survive in this particular soil, while other strains exhibit poor survival and/or may be difficult to detect when present in low numbers.     

A method for the selective isolation ofMyxococcus directly from soil

A new method is described for the selective isolation of species ofMyxococcus directly from soil by dilution plating. The method involves suppression of competing microorganisms with antibiotics combined with air drying and wet heat treatment of soils. Fungi were eliminated by supplementing the plating medium with cycloheximide and nystatin. Non-sporulating bacteria were controlled by air drying soils and then heating aqueous soil dilutions for 10 min at 56°C. The predominant sporulating bacteria in soil,Streptomyces andBacillus, were suppressed by adding either tiacumicin B, ristocetin or vancomycin to the medium. Swarming ofMyxococcus colonies was controlled with a casein digest-yeast extract plating medium (CY-C10 agar). Ultrasound treatment of soil suspensions gave the highest number ofMyxococcus colonies in the soils studied, but these cultures could be recovered without ultrasound. Strains ofMyxococccus fulvus, M. xanthus, M. coralloides, M. stipitatus andM. virescens were isolated from soil using this technique. Soils examined yielded one or twoMyxococcus species per sample.     

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content

Aims: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. Methods and Results: The four methods comprised of 15 isolation steps using selective enrichments (irgasan–ticarcillin–potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25°C. Salmonella–Shigella‐desoxycholate–calcium chloride agar, cefsulodin–irgasan–novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre‐enrichment step with further selective enrichment showed the highest sensitivities (55–66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Conclusions: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. Significance and Impact of the Study: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.     

The respiration of sclerotia of Sclerotium rolfsii

Summary The respiration of sclerotia ofS. rolfsii was investigated using the Warburg constant-volume respirometer to measure oxygen uptake. The effects of age of sclerotia, pH, and temperature were studied. Sclerotia produced on prune agar were ideal for respirometric studies, being uniformly round and of approximately equal size. On a dry weight basis, the respiration rates of sclerotia were considerably less than those of vegetative mycelium. Sclerotia showed a decrease in respiration with increasing age. This was accompanied by morphological changes in the outer hyphal rind of the sclerotium during maturation. The respiration rate of sclerotia was approximately the same at 30° and 40° C, but was significantly lower at 45° C. Respiration of sclerotia was not markedly affected by normally encountered hydrogen-ion concentrations. However, a pH of 8.0 markedly repressed oxygen uptake. Sclerotia produced in rye grain cultures were chemically analyzed. The nitrogen content was 4.7 %, the petroleum-ether-soluble lipid content was 0.7 %, and the crude glycogen content was 14.2 % of the oven dry weight of the sclerotia.Contribution No. 345 from The Department of Botany. Portion of a thesis presented by the senior author in partial fullfillment for the M.S. degree.     

Influence of Nitrogen Fertilizers on Growth, Spore Production and Germination, and Biocontrol Potential of Trichoderma and Gliocladium

The mycelial weight of eight out of nine isolates of Trichoderma spp. and Gliocladium virens increased in media supplemented with 2000 mg/l of nitrogen (N) from the fertilizers NH₄Cl, NaNO₃, and a commercial 20–20–20. In general, the greatest increase in growth (up to 311 %) occurred with 20–20–20. The extent of growth was similar with either NH₄Cl or NaNO₃, but was less than that with 20–20–20. Measured by radial development on agar surfacesgrowth of isolates was either not affected or was constricted by supplemental fertilizers. Production of conidia by six out of eight isolates was stimulated by 20–20–20 but not by NH₄Cl or NaNO₃. Germination of conidia of all isolates, generally was high (> 85 %) on amended and nonamended agar. Chlamydospore formation by three Trichoderma isolates in liquid media was not affected by fertilizers. Antagonism or overgrowth of the pathogen Rhizoctonia solani by Trichoderma isolates in culture was reduced appreciably by NaNO₃, but was not affected by NH₄Cl or 20–20–20. Addition of 20–20–20 to natural soil did not reduce further the survival of R. solani caused by germling preparations of six out of seven Trichoderma isolates. However, reduction in survival of the pathogen caused by a T. hamatum isolate was stimulated further (45 %) by the fertilizer.     

The sclerotia of Coprinus lagopus

Summary Sclerotia of Coprinus lagopus were produced in the laboratory and observed microscopically from initiation to maturity. Isolated bulbous hyphal cells appeared below or on the agar surface as the primary sclerotial initials. Short interhyphal cells had expanded lateral walls and increased in number at their isolated locations, and were surrounded by a thick network of normal mycelium. Increasing in numbers, the bulbous cells developed a tightly compact colony devoid of agar if submerged below the agar surface and lacked normal hyphal filaments. The outer cells thickened formed cell lumina and developed as a protective rind to the mature sclerotium.     

THE EFFECT OF SOME PHYSIOLOGICAL AND ENVIRONMENTAL FACTORS ON SCLEROTIAL ASPERGILLI

Rudolph , Emanuel D. (Ohio State U., Columbus.) The effect of some physiological and environmental factors on sclerotial Aspergilli. Amer. Jour. Bot. 49(1): 71–78. Illus. 1962.—The effect of varying conditions of carbon-nitrogen balance, temperature, pH, and light upon the formation of sclerotia by 6 species of Aspergillus (A. alliaceus, A. avenaceus, A. flavus, A. quercinus, A. sclerotiorum and A. wentii) was studied. On Czapek's agar, optimal growth as well as maximum production of sclerotia and conidia took place at high sucrose and nitrate concentrations. In general, fewer sclerotia were formed with glucose than with sucrose, and very poor growth took place with lactose. Sclerotia were formed best at temperatures that were optimal or below optimal for mycelial growth. The ranges of pH through which sclerotia were formed were narrower than those through which conidia and mycelia were formed. Light had no effect upon sclerotium formation. The formation of sclerotia in A. alliaceus was found to represent the strand-type development. A number of UV-induced strains and a spontaneous mutant strain of A. alliaceus showing varying amounts of sclerotium and conidium production are characterized. It is suggested that the sclerotia in Aspergillus are sterile stromata.     

Empirical evidence of a convection–diffusion model for pH patterns in the rhizospheres of root tips

A recently formulated convection–diffusion model predicted that root growth plus diffusion of protons in the neighbouring soil would lead to particular pH patterns around the moving root tip. To test the predictions of this theory, pH was measured at differing radial distances from the root surface after 24 h of growth in a medium with low diffusivity (sandy soil) and after a shorter period (55 min of growth) in a medium with high diffusivity (agar). In agreement with the theory, the growth zone was found to influence the pH of the soil for distances less than 1 mm from the root surface (even after many hours) and the pH of the agar for a distance of at least 5 mm (after only 1 h). The axial pattern of pH along the surface of soil‐grown Zea mays L. root tips was found to be the same for roots growing at different rates under different temperatures (2·23  mm h^?1 at 26 °C or 1·27 mm h^?1 at 20 °C). Thus, the plant can synchronize proton flux with growth to maintain a particular surface pH pattern within the growth zone. This implies that root tips growing at different rates in response to different temperatures can carry the same microenvironment of pH through a homogeneous soil.     

Soil fungistasis andSclerotium cepivorum berk

Summary Sclerotia ofSclerotium cepivorum Berk. when transferred from staled culture or from soil, were shown to be capable of growth in sterile distilled water. The fact that these propagules germinate readily in sterile conditions without the addition of nutrients indicate that antibiosis is one way by which fungi are maintained in a dormant condition in soil.     

Effect of Bacterial Antagonist on Phytopthora cactorum and Apple Crown Rot

Bacterial antagonist B8 produced an inhibition zone with each of four Phytophthora cactorum isolates on corn meal agar (CMA) plates. Infections with three P. cactorum isolates were significantly reduced when the soil was simultaneously inoculated with B8. Growth of P. cactorum was completely inhibited on CMA amended with 40–100 per cent 10 fold concentrated B8 extract. Percent oospore germination of P. cactorum was significantly reduced when B8 was present in suspension for 9, 12 and 15 days from inoculation. Survival of oospores was significantly reduced at 60 and 90 mm depths in soil. Bacterial antagonist B8 significantly reduced the population of viable P. cactorum oospores in the top 30 mm of soil where oospores generally survive.