Populations of Rhizoctonia solani in soil under crops in rotation with sugar beet

Using a soil debris isolation method, populations of Rhizoctonia solani were monitored over a 4 -yr period in four fields which were initially cropped to sugar beet and in which four areas of Rhizoctonia crown rot diseased beets (DA) and four areas of apparently healthy beets (AH) had been selected and precisely located. Soil from these areas was assayed during the subsequent crops, which included sugar beet, tomato, cucumber, maize and soybean. No significant differences in colony counts were found between the soils in DA and AH on any of 30 sampling dates. R. solani population counts were, in general, quite low, except under sugar beet and following tomato harvest. Areas of diseased beet and high R. solani soil populations that developed in subsequent sugar beet crops did not necessarily coincide with the previously selected diseased areas. High R. solani populations developed from parasitic activity on sugar beet or saprophytically on tomato crop residues. Of the other crops, both maize and soybean may have slightly increased the low R. solani residual populations in soil. The monitoring of R. solani populations in the season prior to, and during the early season of sugar beet cropping did not provide a basis for forecasting disease in fields or sites within fields. The initiation of disease patches in these sugar beet fields was therefore governed by factors other than inoculum density.     

Suppression of Rhizoctonia solani diseases of sugar beet by antagonistic and plant growth-promoting yeasts

AIMS: Isolates of Candida valida, Rhodotorula glutinis and Trichosporon asahii from the rhizosphere of sugar beet in Egypt were examined for their ability to colonize roots, to promote plant growth and to protect sugar beet from Rhizoctonia solani AG-2-2 diseases, under glasshouse conditions. METHODS AND RESULTS: Root colonization abilities of the three yeast species were tested using the root colonization plate assay and the sand-tube method. In the root colonization plate assay, C. valida and T. asahii colonized 95% of roots after 6 days, whilst Rhod. glutinis colonized 90% of roots after 8 days. Root-colonization abilities of the three yeast species tested by the sand-tube method showed that roots and soils attached to roots of sugar beet seedlings were colonized to different degrees. Population densities showed that the three yeast species were found at all depths of the rhizosphere soil adhering to taproots up to 10 cm, but population densities were significantly (P < 0.05) greater in the first 4 cm of the root system compared with other root depths. The three yeast species, applied individually or in combination, significantly (P < 0.05) promoted plant growth and reduced damping off, crown and root rots of sugar beet in glasshouse trials. The combination of the three yeasts (which were not inhibitory to each other) resulted in significantly (P < 0.05) better biocontrol of diseases and plant growth promotion than plants exposed to individual species. CONCLUSIONS: Isolates of C. valida, Rhod. glutinis and T. asahii were capable of colonizing sugar beet roots, promoting growth of sugar beet and protecting the seedlings and mature plants from R. solani diseases. This is the first successful attempt to use yeasts as biocontrol agents against R. solani which causes root diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts were shown to provide significant protection to sugar beet roots against R. solani, a serious soil-borne root pathogen. Yeasts also have the potential to be used as biological fertilizers.     

Chemical techniques for control of stem canker and black scurf (Rhizctonia solani) disease of potatoes

Thiabendazole, iprodione and benodanil were tested for control of stem canker and black scurf disease of potatoes using seed tubers with or without black scurf planted in soil infested or not with Rhizoctonia solani . Dormant seed tubers were treated with fungicide dusts, dips or sprays and before planting dusts were applied to soil or to sprouts on seed tubers.
Plants grown from seed tubers with black scurf had more stem canker, stolon infection and Thanatephorus cucumeris and progeny tubers had more black scurf than from seed without black scurf. Infesting soil with R. solani increased black scurf but not stem canker or stolon infection.
Fungicide treatment of seed tubers controlled all phases of the disease and sprout and soil treatments also had significant effects. Numbers of tubers in July were usually increased by treatment although by harvest many small tubers (>3 cm) had been resorbed. Fungicide treatment substantially increased yield only when shoot tips were pruned by R. solani .
Regression analyses showed that stolon pruning, Thanatephorus and black scurf were significantly related to stem canker, and numbers of tubers in July but not in September were inversely related to stolon pruning.
The significance of sources of inoculum and the effects of controlling disease on tuber production are discussed in relation to methods of fungicide use.     

Stilbella aciculosa: A Potential Biocontrol Fungus against Rhizoctonia solani

A semi‐solid fermentation product of the potential biocontrol fungus Stilbella aciculosa was formulated on wheat bran: water (1:1, w/w) and incubated 5, 10 and 15 days before addition to soil infested with the pathogen Rhizoctonia solani. Generally, preparations did not reduce survival of the pathogen in infested beet seed but they did prevent saprophytic growth of the pathogen from beet seed into soil. The magnitude of reduction by the 15‐day‐old inoculum was greater than that by the 5‐day‐old inoculum. Ten‐day‐old bran preparations of S. aciculosa at rates of 0.5 and 1.0% (w/w) in soil prevented post‐emergence damping‐off of cotton, radish and sugar beet in the glasshouse and a rate of 1.0% gave stands similar to those in the non‐infested control soil. The antagonist, grown on perlite formulated with molasses, cornmeal, alfalfa tissue or corn stover, prevented damping‐off of cotton in a naturally infested soil. However, the stands were not as great as that in soil planted with pentachloronitrobenzene (PCNB)‐treated seed. Toxic metabolites, produced by S. aciculosa developing on various substrates, slightly inhibited the growth of R. solani in culture and induced cytoplasmic leakage of the pathogen mycelium.     

Processed Manure as Carrier to Introduce Trichoderma harzianum: Population Dynamics and Biocontrol Effect on Rhizoctonia solani

Manure pellets produced from processed swine faeces can be used as carrier material for the biocontrol fungus Trichoderma harzianum. The antagonist can grow and sporulate on the processed manure powder as the sole source of carbon and nutrients. The incorporation of conidia in pellets of the processed manure was shown to be feasible on a laboratory scale. Survival of the fungus in the pellets during storage was satisfactory. The population dynamics of T. harzianum were studied using a benomyl-resistance marker after introduction of conidia into soil. The antagonist could colonize and spread through a number of non-sterile soils and was able to establish a stable population over a period exceeding 125 days. Under sterile conditions, the propagation of T. harzianum in soil was much greater than under non-sterile conditions. The incorporation of antagonist conidia in pellets was found to be essential for the successful colonization of non-sterile soil. In growth chamber experiments, application of T. harzianum via processed manure pellets reduced damping-off of sugar beet seedlings caused by Rhizoctonia solani in artificially and naturally infested soil. In artificially infested soil, T. harzianum reduced the population of R. solani and protected beet seedlings from damping-off 3 weeks after introduction. The application of T. harzianum to naturally infested soil increased the number of healthy beet seedlings more than two-fold.     

The Relationship of Plant Age,Soil Temperature,and Population Density of Heterodera schachtii on the Growth of Sugarbeet

There were direct relationships between inoculum density of Heterodera schachtii Schm. (nematode population density), initial soil temperature, the growth of sugarbeets in the greenhouse under controlled temperatures, and nematode populations. Heterodera schachtii was least pathogenic on plants inoculated at 6 wk of age and most pathogenic on plants grown from inoculated germinated seed (0 wk of age). In the field, H. schachtii was least pathogenic on sugarbeets grown at an initial soil temperature of 6 C and most pathogenic on those grown at an initial soil temperature of 24 C. The growth period for sugarbeets at the different soil temperatures was determined by heat units; since penetration of sugarbeet roots by H. schachtii larvae is accelerated at soil temperatures above 10 C, each hour-degree ahove 10 C was counted as one effective heat unit (HU). Using this guideline it was determined that root weight depressions in the greenhouse, for each degree-unit population (HU-UP) where unit population = one larvae/g soil, were 0.052, 0.09, 0.12, and 0.17 mg at initial soil temperatures of 6, 12, 18, and 24 C, respectively. Root weight depressions were 0.28, 0.23, 0.15, and 0.086 mg when plants were inoculated at 0, 2, 4, and 6 wk of age.     

Biocontrol of Damping-off Diseases Caused by Rhizoctonia solani and Pythium ultimum with Alginate Prills of Gliocladium virens, Trichoderma hamatum and Various Food Bases

Alginate prills were formulated with the biomass of isolates of Gliocladium virens and Trichoderma spp. and various food bases (wheat bran, corn cobs, peanut hulls, soy fiber, castor pomace, cocoa hulls and chitin). Alginate prills with G. virens (Gl-21) biomass and all food bases except cocoa hull meal significantly reduced the damping-off of zinnia in a soil-less mix caused by Rhizoctonia solani and Pythium ultimum. The prills with bran, soy fiber, castor pomace or chitin resulted in stands similar to those in the non-infested control. In soil, prills with all the food bases and Thrichoderma hamatum (TRI-4) biomass controlled the damping-off of cotton caused by R. solani and gave stands comparable to, or better than, those in the non-infested control soil. Prills with all the food bases resulted in a proliferation of Gl-21 in a soil-less mix and of Gl-21 and TRI-4 in soil. Prills with food bases and TRI-4 biomass reduced the survival of R. solani in infested beet seed to less than 30%, with bran and chitin being the most effective food bases; prills with Gl-21 biomass and all food bases also reduced the survival of R. solani in beet seed, but not as much as did prills with TRI-4 biomass. In prills containing wheat bran, soy fiber or chitin, the biocontrol isolate Th-58 (T. harzianum) was almost as effective as TRI-4, but isolate Gl-3 (G. virens) was less effective. There was no significant interaction between the biocontrol fungus and the food base. The results suggest that the intrinsic properties of a selected fungus isolate are more important than some formulation variables in biocontrol.     

Soil suppressiveness to Rhizoctonia solani and microbial diversity

Rhizoctonia solani anastomosis group 2-2IIIB causes damping-off, black root rot and crown rot in sugar beet (Beta vulgaris). Based on experiences of growers and field experiments, soils can become suppressive to R. solani. The fungus may be present in the soil, but the plant does not show symptoms. Understanding the mechanisms causing soil suppressiveness to R. solani is essential for the development of environmentally friendly control strategies of rhizoctonia root rot in sugar beet. A bioassay that discriminates soils in their level of disease suppressiveness was developed. Results of bioassays were in accordance with field observations. Preliminary results indicate an active role of microbial communities. Our research is focused on the disentanglement of biological mechanisms causing soil suppressiveness to R. solani in sugar beet. Therefore, we are handling a multidisciplinary approach through experimental fields, bioassays, several in vitro techniques and molecular techniques (PCR-DGGE).     

Analysis of Expression of a Phenazine Biosynthesis Locus of Pseudomonas aureofaciens PGS12 on Seeds with a Mutant Carrying a Phenazine Biosynthesis Locus-Ice Nucleation Reporter Gene Fusion

A derivative of Pseudomonas aureofaciens PGS12 expressing a promoterless ice nucleation gene under the control of a phenazine biosynthesis locus was used to study the expression of a phenazine antibiotic locus (Phz) during bacterial seed colonization. Seeds of various plants were inoculated with wild-type PGS12 and a PGS12 ice nucleation-active phz:inaZ marker exchange derivative and planted in soil, and the expression of the reporter gene was monitored at different intervals for 48 h during seed germination. phz gene expression was first detected 12 h after planting, and the expression increased during the next 36-h period. Significant differences in expression of bacterial populations on different seeds were measured at 48 h. The highest expression level was recorded for wheat seeds (one ice nucleus per 4,000 cells), and the lowest expression level was recorded for cotton seeds (one ice nucleus per 12,000,000 cells). These values indicate that a small proportion of bacteria in a seed population expressed phenazine biosynthesis. Reporter gene expression levels and populations on individual seeds in a sample were lognormally distributed. There was greater variability in reporter gene expression than in population size among individual seeds in a sample. Expression on sugar beet and radish seeds was not affected by different inoculum levels or soil matric potentials of -10 and -40 J/kg; only small differences in expression on wheat and sugar beet seeds were detected when the seeds were planted in various soils. It is suggested that the nutrient level in seed exudates is the primary reason for the differences observed among seeds. The lognormal distribution of phenazine expression on seeds and the timing and difference in expression of phenazine biosynthesis on seeds have implications for the potential efficacy of biocontrol microorganisms against plant pathogens.     

The dynamics of Rhizobium leguminosarum biovar trifolii introduced into soil as determined by immunofluorescence and selective plating techniques

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into a loamy sand and a silt loam, high recovery percentages were determined using quantitative immunofluorescence. Soil type, but not inoculum density between 10⁴ and 10⁸ cells per gram of soil, significantly influenced the recovery percentage of the immunofluorescence technique. Recovery percentages determined using selective plating were independent of either soil type or inoculum density and exceeded those determined by immunofluorescence.
The serological and genetic markers used for detection were stable during 55 days of incubation in phosphate-buffered saline and soil extract solution. After the introduction of R. leguminosarum biovar trifolii into both sterilized soil types, the population increased to 0.5–1×10⁹ cells per gram of soil, but a decline was demonstrated in non-sterile loamy sand and silt loam during incubation of 90 days at 15°C. Starvation of rhizobial cells in the phosphate-buffered saline and soil extract solution, as well as incubation in both soil types, resulted in a significant decrease in mean cell size.     

Colonization of cotton roots by soil micro-organisms

An experiment was carried out to study the colonization of soil microorganisms on cotton roots buried in the soil.Fusarium culmorum, F. solani, F. oxysporum, Macrophomina phaseoli andBacillus sp. were found to be the predominant colonizers followed byBotryodiplodia theobromae, Fusarium acuminatum, Aspergillus niger, A. flavus, Stachybotrys atra, Tilachlidium humicola andRhizopus sp. The practice of leaving cotton roots in the soil would therefore add to the building up of the inoculum potential of the soil inhabiting fungi several of which are serious root pathogens.     

Petroleum-degrading microbial numbers in rhizosphere and non-rhizosphere crude oil-contaminated soil

Phytoremediation can be a cost-effective and environmentally acceptable method to clean up crude oil-contaminated soils in situ. Our research objective was to determine the effects of nitrogen (N) additions and plant growth on the number of total hydrocarbon (TH)-, alkane-, and polycyclic aromatic hydrocarbon (PAH)-degrading microorganisms in weathered crude oil-contaminated soil. A warm-season grass, sudangrass (Sorghum sudanense (Piper) Stapf), was grown for 7 wk in soil with a total petroleum hydrocarbon (TPH) level of 16.6 g TPH/kg soil. Nitrogen was added based upon TPH-C:added total N (TPH-C:TN) ratios ranging from 44:1 to 11:1. Unvegetated and unamended controls were also evaluated. The TH-, alkane-, and PAH-degrading microbial numbers per gram of dry soil were enumerated from rhizosphere and non-rhizosphere soil for vegetated pots and non-rhizosphere soil populations were enumerated from non-vegetated pots. Total petroleum-degrading microbial numbers were also calculated for each pot. The TH-, alkane-, and PAH-degrading microbial numbers per gram of dry soil in the sudangrass rhizosphere were 3.4, 2.6, and 4.8 times larger, respectively, than those in non-rhizosphere soil across all N rates. The presence of sudangrass resulted in significantly more TH-degrading microorganisms per pot when grown in soil with a TPH-C:TN ratio of 11:1 as compared to the control. Increased plant root growth in a crude oil-contaminated soil and a concomitant increase in petroleum-degrading microbial numbers in the rhizosphere have the potential to enhance phytoremediation.     

Soil receptiveness to VA mycorrhizal association: Concept and method

The concept of soil receptiveness widely used for soil borne pathogens, is applied to the fungi forming vesicular-arbuscular endomycorrhizae. The authors propose a method for determining the mycorrhizal soil receptiveness (MSR) using leek, a highly mycotrophic plant, as a host for a bioassay. Under controlled conditions, populations of leek plants are grown in a soil inoculated with a range of inoculum levels. The inoculum consists of standardized root pieces infected with G. intraradices which are considered as propagules. The relationship between the percentage of plants forming mycorrhizae and the level of inoculum is used as a basis for determining the quantity of inoculum required to obtain mycorrhizae formation on 50% of the host plant population. The results are defined in terms of MSR unit, and are expressed as number of propagules corresponding to a MSR₅₀ unit, or as MSR₅₀ unit per propagule. This method is illustrated in a comparative study of four agricultural soils from France.     

Genes involved in cyclic lipopeptide production are important for seed and straw colonization by Pseudomonas sp. strain DSS73

Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.     

Fungal inoculum properties and its effect on growth and enzyme activity of Trametes versicolor in soil

The effect of fungal inoculum properties on colonization of nonsterile soil by three isolates of the white-rot fungus Trametes versicolor was investigated. Fungal inoculum properties were examined in separate experiments and were fungal inoculum composition, age of fungal inoculum, concentration of the inoculum and inoculation method. The fungal inoculum composition study compared pine versus poplar sawdust as the basic carrier with varying amounts of corn grit, corn meal and starch. The age of the fungal inoculum studied ranged from 3 to 21 days. The inoculum concentration gradually increased from 0 to 50% (v/v). The study assessing inoculation method compared mixing with layering techniques. The effect of moisture conditions of soil, sawdust and sand in combination with two inoculation methods (mixing versus point source inoculation) on colonization by T. versicolor was also determined. Colonization of soil was always assessed visually and enzymatically monitoring mycelial growth, biological potential (fluorescein diacetate assay) and laccase levels. Generally, the three different assessment methods correlated (P < 0.05) with each other. A fungal inoculum based on pine sawdust supported white-rot fungal growth in soil better than a poplar sawdust basis. Colonization of soil by T. versicolor was improved by increasing the corn content of the fungal inoculum. Younger (<7 days old) fungal inoculum resulted in better soil colonization than older (>10 days). A strong correlation (P < 0.001) was observed between the amount of fungal inoculum used in the soil augmentation and white-rot fungal colonization of soil. Inoculation of the fungal inoculum into soil by mixing was preferable over application in layers or point source inoculation. Moisture level did not influence biological potential measurements, but affected mycelial growth and laccase expression.     

Structure, production characteristics and fungal antagonism of tensin - a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578

AIM: To study the antagonistic activity by Pseudomonas fluorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani. METHODS AND RESULTS: Strain 96.578 produced a new cyclic lipopeptide, tensin. High tensin production per cell was detected in liquid media with glucose, mannitol or glutamate as growth substrate while fructose, sucrose and asparagine supported low production. Tensin production was nearly constant in media with different initial C levels, while low initial N contents reduced production. When applied to sugar beet seeds, strain 96.578 produced tensin during seed germination. When challenged with strain 96.578 or purified tensin, Rhizoctonia solani reduced radial mycelium extension but increased branching and rosette formation. CONCLUSION: The antagonistic activity of strain 96.578 towards Rhizoctonia solani was caused by tensin. SIGNIFICANCE AND IMPACT OF THE STUDY: When coated onto sugar beet seeds, tensin production by strain 96.578 could be of significant importance for inhibition of mycelial growth and seed infection by Rhizoctonia solani.     

Viable but non-culturable salmonellas in soil

P.E. TURPIN, K.A. MAYCROFT, C.L. ROWLANDS AND E.M.H. WELLINGTON. 1993. An enzyme-linked immunosorbent assay (ELISA) and a microwell fluorescent antibody (FA) direct count method have been developed for the monitoring of salmonellas in soil. Both methods have a minimum detection level of ca 10⁶ cells per gram of soil. The FA direct count method gave a linear recovery for the inoculum range 10⁶–10⁹ cells per gram of soil. When monitored by plate counts the survival of salmonellas was greater in a sterile than in a non-sterile soil. Evidence was found for the production of viable but non-culturable salmonellas in non-sterile soil; plate counts dropped rapidly with time, but FA direct counts and ELISA remained level. The salmonella cells became progressively smaller and rounder with time. Dead salmonella cells introduced into soil rapidly disappeared.     

Nodulation of lucerne (Medicago sativa L.) in an acid soil: Effects of inoculum size and lime-pelleting

The effects of inoculum level and lime-pelleting were studied in an acid soil with respect to the nodulation and growth of lucerne (Medicago sativa cv Resis) and the population dynamics of Rhizobium meliloti. In small root-boxes (rhizotrons), the in-situ survival of inoculated rhizobia was studied in the micro-environment around the seed for a period of 12 days after sowing. During the initial 24 hours, a strong increase in rhizobial numbers was measured, concomitantly with the development of roots. As a result of lime-pelleting, rhizobial numbers were higher only at 3 days after sowing (P<0.05). Later, this difference diminished steadily. Addition of lime did not increase the adhesion of the rhizobia to the seedling tap root. Plant responses to inoculation were studied in pots. To obtain optimal nodulation, the soil had to be neutralized around the seed with lime and at least 10⁵ cells of R. meliloti were required. With more than 10⁵ rhizobia per seed, lime-pelleting increased the number of crown-nodulated seedlings from 24% to 77%. Higher numbers of rhizobia could not compensate the effect of lime. A strong correlation was found between crown nodulation, nitrogen content and dry weight of the shoots.     

Estimation of the number of polyhydroxyalkanoate (PHA)-degraders in soil and isolation of degraders based on the method of most probable number (MPN) using PHA-film.

A new method to estimate the number of polyhydroxyalkanoates (PHA)-degraders in soil and to isolate degraders, called the film-MPN method, is proposed. The incubation time was measured by the first order reaction (FOR) model. This method was used to estimate numbers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)[P(3HB-co-3HV)]- and poly(3-hydroxyvalerate-co-4-hydroxybutyrate)[P(3HB-co-4HB)]-degraders in garden soil (4.30 x 10(5) and 2.15 x 10(5) aerobic degraders per gram of dry soil, respectively). The number of P(3HB-co-3HV)-degraders in paddy field soil was 5.06 x 10(5) aerobic degraders per gram dry soil. Also, several P(3HB-co-3HV)-degraders were isolated directly from positive-growth tubes of high dilution.     

Study of the aggressiveness of Rhizoctonia solani isolates

This paper presents an in vitro test to screen the pathogenicity of different Rhizoctonia solani isolates on a host range. The level of aggressivity of the different isolates was different for several host plants tested. There were significant differences between the crops and the isolates tested. In general, the disease level was higher on beans, lettuce and cabbage. In carrot and rye grass the level of infection was lower for the isolates of R. solani tested. The potato isolates of R. solani were less aggressive than the isolates coming from maize, fodder beet and sugar beet. The R. solani isolates were also biochemically characterized by pectic zymograms: the isolates Rs0401 (from maize) and Rs0504 (from sugar beet) belong both to the anastomosis group AG2-2.