Soil receptivity toFusarium solani f. sp.pisi and biological control of root rot of pea

Potential antagonists ofFusarium solani f. sp.pisi (Fsp) were selected from soil samples with varying degrees of receptivity to this pathogen. They were tested against Fsp isolate 48 (Fs48), in increasingly complex systems. Most species testedin vitro were able to antagonize Fs48. No relation could be establishedin vitro between the receptivity of the soil from which an isolate originated and its antagonism to Fs48. In soils naturally infested with pea root rot pathogens, which were stored humid at 4°C for a period longer than a year, various isolates ofFusarium, Gliocladium andPenicillium spp. were able to reduce root rot. After sterilization of these soils, onlyGliocladium roseum isolates, added at 10⁵ conidia g^–1 dry soil, significantly reduced disease severity and prevented root weight losses caused by Fs48 at 10⁴ conidia g^–1 dry soil. In soils in which the biota were activated by growing peas before the assays, doses of 10⁶ and 10⁷ ofG. roseum were required to reduce root rot. In these soils, the antagonistic effects of fluorescent pseudomonad strains from soil of low receptivity to Fsp were variable. Some strains of fluorescent pseudomonads, from soil moderately receptive to Fsp and from highly infested soils, were also able to reduce root rot. Disease suppression by pseudomonad strains was more evident in the absence than in the presence ofAphanomyces euteiches in the root rot pathogen complex. The role of receptiveness of the soil with regard to potential antagonists is discussed.     

Postinfection Biological Control of Oomycete Pathogens of Pea by Burkholderia cepacia AMMDR1

ABSTRACT Burkholderia cepacia AMMDR1 is a biocontrol agent that reduces Pythium damping-off and Aphanomyces root rot severity on peas in the field. We studied the effect of B. cepacia AMMDR1 on post-infection stages in the life cycles of these pathogens, including mycelial colonization of the host, production of oogonia, and production of secondary zoospore inoculum. We used Burkholderia cepacia 1324, a seed and rootcolonizing but antibiosis-deficient Tn5 mutant of B. cepacia AMMDR1, to study mechanisms of biological control other than antibiosis. B. cepacia AMMDR1 significantly reduced Pythium aphanidermatum postinfection colonization and damping-off of pea seeds, even when the bacteria were applied 12 h after zoospore inoculation. B. cepacia AMMDR1 also significantly reduced colonization of taproots by Aphanomyces euteiches mycelium, but only when the bacteria were applied at high population densities at the site of zoospore inoculation. The antibiosisdeficient mutant, B. cepacia 1324, had no effect on mycelial colonization of seeds or roots by Pythium aphanidermatum nor A. euteiches, suggesting that antibiosis is the primary mechanism of biological control. B. cepacia AMMDR1, but not B. cepacia 1324, reduced production of A. euteiches oogonia. This effect occurred even when the population size of B. cepacia AMMDR1 was too small to cause a reduction in lesion length early on in the infection process and may result from in situ antibiotic production. B. cepacia AMMDR1 had no effect on the production of secondary zoospores of A. euteiches from infected roots. The main effects of B. cepacia AMMDR1 on postinfection stages in the life cycles of these pathogens therefore were reductions in mycelial colonization by Pythium aphanidermatum and in formation of oogonia by A. euteiches. No mechanism other than antibiosis could be identified.     

Hierarchical Analysis of Diversity, Selfing, and Genetic Differentiation in Populations of the Oomycete Aphanomyces euteiches

ABSTRACT Relatively little is known about the population biology of the legume pathogen Aphanomyces euteiches. A. euteiches is a soilborne pathogen causing Aphanomyces root rot of several legumes, including alfalfa, bean, lentil, and pea. Our objectives were to assess the degree of diversity, selfing, and population differentiation in A. euteiches. We contrasted populations within and among two geographically separated fields with a history of pea production. Molecular genotyping relied on amplified fragment length polymorphism analysis. Samples of A. euteiches recovered from two fields in northeast Oregon and western Washington confirmed previous reports of moderately high genetic diversity in populations of A. euteiches at the regional scale, but revealed higher-than-expected genotypic diversity within individual soil samples. Populations of A. euteiches were significantly differentiated at the soil sample, field, and regional level. The population structure appears to be patterned by regular selfing via oospores, a mixed reproductive system including both asexual and sexual reproduction, with occasional migration of novel genotypes or outcrossing.     

Bioassay to assess root rot in pea and effect of root rot on yield

Infection of pea roots by soil-borne pathogens causes foot and root rot. In 1985 research was started to develop a method to predict the root rot likely to occur in prospective pea fields. In a bioassay the pea cultivar Finale was sown in a composite soil sample from each field in pots under standardized conditions in the greenhouse. The plants were removed at the green bud stage and the severity of root rot recorded. Between 1985 and 1988 approximately 200 field pea crops were monitored for root rot development. Forty-eight fields were bioassayed in 1986, 51 in 1987 and 30 in 1988. Each year, root rot readings in the bioassay and disease severity readings at field sampled plants at flowering and green pod were linearly correlated (P<0.001). As the degree of root rot in the field crop increased, there was a proportional lower yield. In heavily infested fields, up to a 50% yield reduction occurred.The bioassay in pots proved to be a reliable method for predicting root rot severity in sampled pea fields.     

Effect of Temperature on and Histopathology of the Interaction Between Meloidogyne incognita and Thielaviopsis basicola on Cotton

ABSTRACT Controlled environments were used to study the relationship between the root-knot nematode (Meloidogyne incognita) and Thielaviopsis basicola on cotton. Temperature treatments were continuous 20, 24, and 28 degrees C or two cyclic linear regimes with ranges of 14 to 32 or 18 to 28 degrees C over 24 h. Cotton seeds were planted in fumigated soil infested with T. basicola, M. incognita, or both. After 42 days, pathogen effects on plant growth and pathogen development were evaluated. Histology was conducted on roots collected 14, 28, and 42 days after planting in the continuous 24 degrees C treatment. Reductions in plant height-to-node ratio and total fresh weight were observed for soils infested with both pathogens compared with the control or with soils infested with either pathogen, except for M. incognita-infested soil at 28 degrees C. T. basicola reduced root galling and reproduction of the nematode at all temperatures. Vascular discoloration caused by T. basicola was greater in the presence of M. incognita compared with that by T. basicola alone. At 2 and 4 weeks, histological studies showed that plants grown in all T. basicola-infested soils contained chlamydospore chains on the root surface and in cortical cells. The fungus was not observed inside the vascular cylinder. Roots from 4-week-old plants from soils infested with T. basicola and M. incognita showed fungal sporulation in vascular tissue and localized necrosis of vascular tissue adjacent to the nematodes. At 6 weeks, plants grown in soil infested with T. basicola alone exhibited no remaining cortical tissue and no evidence of vascular colonization by the fungus. Six-week-old plants grown in T. basicola + M. incognita-infested soils exhibited extensive vascular necrosis and sporulation within vascular tissue. These studies suggest that coinfection expands the temperature ranges at which the pathogens are able to cause plant damage. Further, M. incognita greatly increases the access of T. basicola to vascular tissue.     

Biological Control of Pathogens Causing Root Rot Complex in Field Pea Using Clonostachys rosea Strain ACM941

ABSTRACT Pea root rot complex (PRRC), caused by Alternaria alternata, Aphanomyces euteiches, Fusarium oxysporum f. sp. pisi, F. solani f. sp. pisi, Mycosphaerella pinodes, Pythium spp., Rhizoctonia solani, and Sclerotinia sclerotiorum, is a major yield-limiting factor for field pea production in Canada. A strain of Clonostachys rosea (syn. Gliocladium roseum), ACM941 (ATCC 74447), was identified as a mycoparasite against these pathogens. When grown near the pathogen, ACM941 often was stimulated to produce lateral branches that grew directly toward the pathogen mycelium, typically entwining around the pathogen mycelium. When applied to the seed, ACM941 propagated in the rhizosphere and colonized the seed coat, hypocotyl, and roots as the plant developed and grew. ACM941 significantly reduced the recovery of all fungal pathogens from infected seed, increased in vitro seed germination by 44% and seedling emergence by 22%, and reduced root rot severity by 76%. The effects were similar to those of thiram fungicide, which increased germination and emergence by 33 and 29%, respectively, and reduced root rot severity by 65%. When soil was inoculated with selected PRRC pathogens in a controlled environment, seed treatment with ACM941 significantly increased emergence by 26, 38, 28, 13, and 21% for F. oxysporum f. sp. pisi, F. solani f. sp. pisi, M. pinodes, R. solani, and S. sclerotiorum, respectively. Under field conditions from 1995 to 1997, ACM941 increased emergence by 17, 23, 22, 13, and 18% and yield by 15, 6, 28, 6, and 19% for the five respective pathogens. The seed treatment effects of ACM941 on these PRRC pathogens were greater or statistically equivalent to those achieved with thiram. Results of this study suggest that ACM941 is an effective bioagent in controlling PRRC and is an alternative to existing chemical products.     

土壤中烟草根黑腐病菌的实时定量PCR检测技术研究

 Thielaviopsis basicola is a soil-borne plant pathogen which causes root rot disease in tobacco plants. Detection and monitoring of T. basicolain soil is of great significance to control this disease. Based on the differences in internal transcribed spacer (ITS) sequences of T. basicola and other fungal pathogens, a specific primer pair Tb1/Tb2 for T. basicolawas developed. The results showed that the primer pair gave a single amplicon of 330 bp from T. basicola and revealed no undesirable cross-reaction with other seven soil-borne pathogen isolates and three tobacco rhizosphere dominant fungi isolates. With a series of 10-fold genomic DNA dilutions of T. basicola, the detection limit of 1 pg/μL in conventional PCRand100 fg/μL in real-time quantitative PCR was achieved. With DNA from the soil inoculated with different numbers of T. basicola conidia, the detection limit was 10 conidia per reaction in conventional PCR and 0.4 conidia per reaction in real-time quantitative PCR.     

Relation between cropping frequency of peas and other legumes and foot and root rot in peas

The relation between the frequency of legume crops in a rotation and the root rot severity in pea was examined in a field survey. Additionally, greenhouse experiments were performed with soil samples from legume rotation trials or from farmers' fields. The frequency of pea crops in current rotations proved to be much less than the recommended value of one in six years. The correlation between pea root rot and the number of years that pea or other legumes were not grown on the field under consideration (called crop interval) was weak. Root rot severity correlated better with the frequency of peas or legumes in general over a period of 18 years, but the frequency still explained only a minor fraction of the variation in disease index. Some experimental data pointed to the occurrence of a highly specific pathogen microflora with continuous cropping of only one legume species, but this phenomenon probably does not occur in farmers' fields. In field samples, root disease index for pea correlated well with that for field bean. The survival of resting structures of pathogens such asAphanomyces euteiches probably explains why the frequency of legume cropping has a higher impact than crop interval on root disease incidence. Pea-free periods and legume frequencies have a poor predictive value for crop management purposes.     

Herbicide Effects on Sugarcane Growth, Pythium Root Rot, and Pythium arrhenomanes

ABSTRACT Six herbicides were evaluated for their effects on Pythium root rot and growth of sugarcane in greenhouse experiments and on in vitro mycelial growth rate of Pythium arrhenomanes. Pendimethalin and atrazine were most inhibitory to mycelial growth, but neither reduced root rot severity. Asulam, atrazine, and metribuzin were not phytotoxic to sugarcane and did not affect root rot symptom severity in clay loam or silt loam field soils. Atrazine and metribuzin increased shoot number, and atrazine increased total shoot weight for treated plants in silt loam soil. Glyphosate, pendimethalin, and terbacil were phytotoxic to sugarcane. These herbicides increased root rot severity, but the extent to which growth reductions resulted from increased disease severity or from direct herbicide injury was not clear. Adverse effects on plant growth and root rot severity were greater in clay loam than in silt loam soil. The results suggest that sugarcane injury from some herbicides is compounded by increased severity of root rot.     

Wheat Genotype-Specific Induction of Soil Microbial Communities Suppressive to Disease Incited by Rhizoctonia solani Anastomosis Group (AG)-5 and AG-8

ABSTRACT The induction of disease-suppressive soils in response to specific cropping sequences has been demonstrated for numerous plant-pathogen systems. The role of host genotype in elicitation of the essential transformations in soil microbial community structure that lead to disease suppression has not been fully recognized. Apple orchard soils were planted with three successive 28-day cycles of specific wheat cultivars in the greenhouse prior to infestation with Rhizoctonia solani anastomosis group (AG)-5 or AG-8. Suppressiveness to Rhizoctonia root rot of apple caused by the introduced isolate of R. solani AG-5 was induced in a wheat cultivar-specific manner. Pasteurization of soils after wheat cultivation and prior to pathogen introduction eliminated the disease suppressive potential of the soil. Wheat cultivars that induced disease suppression enhanced populations of specific fluorescent pseudomonad genotypes with antagonistic activity toward R. solani AG-5 and AG-8, but cultivars that did not elicit a disease suppressive soil did not modify the antagonistic capacity of this bacterial community. When soils were infested prior to the initial wheat planting, all cultivars were uniformly susceptible to R. solani AG-8. However, when pathogen inoculum was added after three growth-cycles, wheat root infection during the fourth growth-cycle varied in a cultivar specific manner. The same wheat cultivar-specific response in terms of transformation of the fluorescent pseudomonad community and subsequent suppression of Rhizoctonia root rot of apple was observed in three different orchard soils. These results demonstrate the importance of host genotype in modification of indigenous saprophytic microbial communities and suggest an important role for host genotype in the success of biological control.     

Reassessment of the Role of Saprophytic Activity in the Ecology of Thielaviopsis basicola

ABSTRACT The ability of Thielaviopsis basicola to survive saprophytically in soil was investigated using root tissue from susceptible hosts as organic substrates. Inoculum densities were lower in soils amended with root tissue than in nonamended controls after 2 and 4 weeks of incubation. The greatest decrease occurred in soils containing the highest concentration of root tissue or in soils in which root tissue included the soluble components of the living root. Reproduction by T. basicola also was examined in axenic media containing either killed root pieces or various carbohydrates as the sole carbohydrate source. T. basicola utilized killed root tissue as a carbohydrate source in axenic media, particularly in cultures in which root tissue included the soluble components. Enzymatic activities of T. basicola, however, did not result in maceration of the root tissue. T. basicola utilized sucrose and cellobiose, but did not utilize structural carbohydrates such as cellulose, hemicellulose, or pectin. Based on the absence of significant saprophytic ability, T. basicola should be classified ecologically as an obligate parasite.     

Pathogenic characteristics of isolates of Aphanomyces euteiches from pea in France

Aphanomyces root rot ( Aphanomyces euteiches ) has become a very destructive disease in French pea crops since 1993. The host specificity of the French pea-infecting populations of this pathogen was investigated by inoculating pea, common vetch, alfalfa, broad bean and green bean with 91 pea-infecting A. euteiches isolates, originating from the main areas of infestation in France. These isolates were compared to 13 isolates from various countries and hosts (pea, green bean, alfalfa). Virulence phenotypes were defined according to the pathogenicity data on the different hosts: all isolates from France infected two to five legume species, with most infecting pea, vetch, alfalfa and broad bean. Four pathotypes were characterized within the French isolates: one type corresponded to broad host range isolates, the second was composed of isolates preferentially agressive on pea/vetch/alfalfa and weakly aggressive on broad bean, and two others corresponding to more specialized isolates that preferentially infected pea/vetch or pea/vetch/alfalfa. Most isolates from France were preferentially pathogenic on pea, like the pea-infecting isolates from other countries, but were less specialized than the alfalfa- and green bean-infecting isolates from other countries. These results suggest that A. euteiches isolates may be maintained on wild or cultivated legumes other than pea in France.     

The occurrence in Denmark of black root rot of pea caused by Thielaviopsis basicola

Thielaviopsis basicola has been shown to be a root pathogen of pea of considerable importance in Denmark. The fungus is only found in fields with one or more previous pea crops in the field history. In the dry and warm growing season of 1989 the fungus was found in 0·6% and 3·2% of the fields in two separate areas in Denmark. In the fields where T. basicola was detected the average disease severity index in plant samples was 51·8, whereas the average disease severity index in plant samples without the fungus was 27·0. The average yield of green peas was reduced from 5167 to 4171 kg/ha when T. basicola was present. For detection and isolation of T. basicola it is important to use a technique combining microscopic examination, a semi-selective medium and a dilution plate method.     

Interactions between the biocontrol agent Pseudomonas fluorescens CHA0 and Thielaviopsis basicola in tobacco roots observed by immunofluorescence microscopy

Pseudomonas fluorescens CHA0 protects plants from damage caused by several soilborne fungi. In this work, immunofluorescence microscopy was used to investigate the colonization of tobacco roots by CHA0 and its physical relationship with the black root rot fungus Thielaviopsis basicola . The pseudomonad colonized the rhizoplane shortly after planting of tobacco seedlings in sterile soil microcosms, in which it had been introduced as soil inoculant. CHA0 was found between and inside cells in the epidermis and the cortex, as well as in the xylem vessels, within 4–7 days after planting of seedlings. The presence of CHA0 delayed the colonization of the interior of tobacco roots by T. basicola compared with the treatment in which only the fungus had been inoculated. Likewise, the pseudomonad reduced the extent of black root rot from 82% to 28%. However, CHA0 was seldom found in contact with the mycelium of T. basicola or in its vicinity, indicating that direct colonization of the mycelium of T. basicola by CHA0 was not required for protection of tobacco against black root rot. Overall, the results suggest that the interior of the root is a key site for implementation of the strain's biocontrol activity against soilborne plant-pathogenic fungi.     

Spatial distribution of Aphanomyces euteiches inoculum in a naturally infested pea field

The main objective of the study was to describe the horizontal and vertical distribution of Aphanomyces root rot in a naturally infested pea field. Measurements of inoculum potential clearly indicated a horizontal distribution of inoculum among several foci in the field, these foci differing in size and disease intensity. A highly significant relationship was observed between disease severity on plants during the cropping season and soil inoculum potential. In terms of the vertical distribution of inoculum in the soil, detection was maximal at a depth of 10 to 40 cm, but inoculum was detected down to a depth of 60 cm. Generally, inoculum potential was lowest for the layers at depths of 0 to 10 and 50 to 60 cm. Inoculum distribution and the value of the methodologies used are discussed in terms of possible use for epidemiology and disease forecasting.     

Disease assessment of pea lines with resistance to foot rot pathogens: protocols for in vitro selection

Cultural conditions were optimized so that disease reactions of plantlets and callus tissue from partially resistant and susceptible genotypes of Pisum sativum could be differentiated when inoculated with Fusarium solani f.sp. pisi. Disease was assessed using both semi-quantitative (disease score/fungal diameter) and quantitative methods (ergosterol assay).
After optimization of inoculum concentration and incubation period it was possible to differentiate the disease reactions of plantlets using disease scores and amount of ergosterol in the tissues.
Incubation period, temperature and the auxin added to the media were important in allowing differentiation of callus tissue cultures. These differences were visible by measurement of the diameter of fungal growth on the callus and the amount of ergosterol in the tissues, but not with disease scores.
The plantlet test was also applicable to the other foot rot pathogens Phoma medicaginis var. pinodella and Aphanomyces euteiches , allowing differentiation of the disease reactions of a partially resistant and susceptible genotype using disease scores. The ergosterol assay could be used to quantify infection caused by P. medicaginis var. pinodella , but this sterol was not detectable in mycelium of A. euteiches. In this fungus the presence of cholesterol was detected, which may be used to quantify infection.     

Incidence of soil-borne plant pathogens isolated from barley and winter wheat, and other crops in the rotation, on Prince Edward Island

Barley and winter wheat were sampled over 3 years to characterize soil-borne organisms involved with cereal crown and root disease complexes. Winter wheat crowns were infected more often by Fusarium avenaceum and F. sambucinum than barley crowns. Bipolaris sorokiniana and F. graminearum were more common in crown tissue of barley underseeded with clover than in winter wheat crowns. In roots, the incidence of F. avenaceum was highest in winter wheat. In soil, populations of Rhizoctonia solani and F. sambucinum were higher in winter wheat than barley underseeded with ryegrass. Stunt nematodes (Tylenchorhynchus spp.) were greatest in winter wheat and barley underseeded with ryegrass. The incidence of F. avenaceum in roots of winter wheat correlated positively with the severity of crown and root rot symptoms. The severity of barley crown and root rot correlated positively with the incidence of R. solani in crowns, F. avenaceum in crowns and roots, B. sorokiniana in roots, and numbers of stunt nematodes in the soil. The incidence of soil-borne organisms was also recorded in annual ryegrass. soybean, potato, pea, and clover which were grown in rotation with barley and winter wheat. The incidence of R. solani and F. avenaceum was highest in clover crown tissue, and F. sambucinum was prevalent in soybean hypocotyls. Soil population levels of R. solani, F. avenaceum , and stunt nematodes were greatest in ryegrass and pea soil just prior to ploughing down or harvesting these crops, respectively.     

Potato brown rot incidence and severity under different management and amendment regimes in different soil types

Ralstonia solanacearum race 3 biovar 2, the causative agent of potato brown rot (bacterial wilt), is an economically important disease in tropical, subtropical and temperate regions of the world. In view of previous reports on suppression of the disease by organic amendments, and the expansion of organic agriculture, it was timely to compare the effects of organic and conventional management and various amendments on brown rot development in different soils (type: sand or clay; origin: Egypt or the Netherlands). Brown rot infection was only slightly reduced in organically compared to conventionally managed sandy soils from Egypt, but organic management significantly increased disease incidence and pathogen survival in Dutch sandy and clay soils, which correlated with high DOC contents in the organic Dutch soils. There was no correlation between disease incidence or severity and bacterial diversity in the potato rhizosphere in differently managed soils (as determined by 16S DGGE). NPK fertilization reduced bacterial wilt in conventional Egyptian soils but not in Dutch soils. Cow manure amendment significantly reduced disease incidence in organic Dutch sandy soils, but did not affect the bacterial population. However, cow manure did reduce densities of R. solanacearum in Egyptian sandy soils, most probably by microbial competition as a clear shift in populations was detected with DGGE in these and Dutch sandy soils after manure amendment. Amendment with compost did not have a suppressive effect in any soil type. The absence of a disease suppressive effect of mineral and organic fertilization in Dutch clay soils may be related to the already high availability of inorganic and organic nutrients in these soils. This study shows that the mechanism of disease suppression of soil-borne plant pathogens may vary strongly according to the soil type, especially if quite different types of soil are used.     

Quantification of Cylindrocarpon destructans f. sp. panacis in soils by real-time PCR

Ginseng ( Panax quinquefolius ) is an important cash crop in various regions of North America, but yields are often reduced by various root pathogens. A quantitative real-time PCR (qPCR) assay for Cylindrocarpon destructans f. sp. panacis (CDP), the cause of a root rot and replant disease which discourages successive cropping of ginseng on the same site, was developed to quantify the levels of this pathogen in soils previously cropped with ginseng. DNA was extracted from 5-g samples of soil. In pasteurized soils which were re-infested with varying levels of the pathogen, qPCR estimates of pathogen DNA were significantly correlated with disease severity ( r  = 0·494) and with counts of colony-forming units ( r  = 0·620) obtained with an agar medium. In several naturally infested field soils, qPCR estimates of CDP-DNA concentration were significantly correlated with disease severity ( r  = 0·765) and these concentrations were estimated to range from 0 to 1·48 ng g⁻¹ dried soil. A principal components analysis did not show any strong relationships between soil chemistry factors and the concentration of pathogen DNA. The approach outlined here allows the quantification of current populations of CDP in soil many years after ginseng cultivation and the prediction of disease severity in future crops. The method should be generally applicable to root diseases of many crops.     

Suppression of Rhizoctonia solani in potato fields. II. Effect of origin and degree of infection with Rhizoctonia solani of seed potatoes on subsequent infestation and on formation of sclerotia

The seed potatoes used in these experiments had been grown in a slightly acid pleistocene sandy soil or in a marine, holocene sandy loam. They were free of sclerotia ofR. solani or lightly or moderately speckled with them. Seed potatoes from the sandy soil produced plants that suffered less fromRhizoctonia than plants from seed potatoes that had been grown on the marine sandy loam. Similarly harvested tubers had, in a non-conducive soil and in conducive soils with a (very) low inoculum density ofR. solani, fewer sclerotia when they came from seed potatoes grown in an acid sand. In each soil, the degree of infestation of the crop not only depended on the severity of infection of the seed potatoes, but also on their origin. With regard to sclerotia production on tubers, three types of soil were distinguished: suppressive, conducive with a high, and conducive with a very low inoculum density ofR. solani. The differences in infestation and in the amounts of sclerotia on tubers between the crop grown from seed potatoes from the sandy soil and that from seed potatoes from the marine sandy loam soil, is attributed to a richer load of antagonists on the former and possibly to a larger proportion of saprophyticRhizoctonia strains among their sclerotia. The antagonists seem to be inhabitants of the subterranean parts of the plant and to function independently of the soil. This implies possibilities for their use in biological control.