Biological control of the take-all fungus, Gaeumannomyces graminis, by Phialophora radicicola and similar fungi

Phialophora radicicola is an avirulent fungal root-parasite of grasses and cereals, with runner hyphae like those of Gaeumannomyces graminis. Weakly and non-pathogenic varieties of these fungi control the pathogens, G. graminis vars. tritici and avenue. Biology of these fungi is considered and the evidence for biological control and possible mechanisms reviewed; control is probably widespread in natural plant communities, and host-mediated, perhaps by induction of plant resistance mechanisms.Prospects for application of biological control seem best for P. radicicola var. graminicola established on grass crops, as this is already exploited in British agriculture. New evidence is presented on the effects of grassland factors on this fungus, especially sward composition, age, mineral nutrition and management practices: its population might often be limited by the rate of new root production to replace those with cortices already colonized. Prospects for control by seed inoculation with P. radicicola var. radicicola and G. graminis var. graminis also seem good, but possible dangers of introducing them into cereal cropping are emphasized. The weak pathogens might be used also for indirect control by establishing hyper-parasites or inducing disease suppression (like take-all decline) in soils, but there is no evidence for ‘Phialophora decline’, at least in well-managed grasslands. Finally, different biocontrols of take-all might be combined, and biological with chemical ones for ophiobolus patch disease of turf.P. radicicola var. graminicola has a slight beneficial effect on grass yield, even when the pathogens, G. graminis vars. tritici and arenae arc absent; this probably contributes to its abundance in natural grasslands in Britain. The scale of biological control by this and similar fungi might explain why, in their absence, effective plant resistance to G. graminis is uncommon in the Gramineae.     

The incidence of Gaeumannomyces graminis var. Tritici and Phialophora radicicola var. Graminicola on wheat grown after different cropping sequences

The effects of three treatment cropping sequences (fallow, lucerne or grass-clover ley) on the incidence of Gaeumannomyces graminis var. tritici and Phialophora radicicola var. graminicola were measured in a field experiment. Increases in G. g. tritici population in the soils of the first wheat crop and the incidence of take-all in the second wheat crop were greater after fallow or lucerne than after grass-clover. These differential increases were not associated with differences in survival of G. g. tritici during the treatment cropping but were correlated negatively with the population of P.r. graminicola in the soil. After the third wheat crop the P. r. graminicola population after grass-clover had decreased and take-all was as prevalent as after fallow or lucerne.     

Colonization of wheat roots by Gaeumannomyces graminis inhibited by specific soils,microorganisms and ammonium-nitrogen

Lineal extension of Gaeumannomyces graminis var. tritici hyphae along roots of intact wheat plants growing in soils was measured. Hyphal growth rates were lower in soils treated with NH₄⁺-N than with NO₃^?-N. In a soil that is suppressive to the take-all disease, the controlling influence of NH₄⁺-N was eliminated by soil fumigation (methyl bromide), and reintroduced to fumigated soil by additions of 1% nonsterile soil. Effects of fumigation on hyphal growth were absent in a nonsuppressive soil, and in NO₃^?-treatments of the suppressive soil. When inocula of selected groups of wheat rhizoplane microflora were reintroduced into a fumigated or a soil-reinoculated soil via a root-food base, the Pseudomonas spp. consistently appeared more suppressive in NH₄⁺-N treatments than the general bacterial flora, Bacillus spp. spores, streptomycetes, and fungi.     

Colonisation of seminal roots of wheat and barley by egg-parasitic nematophagous fungi and their effects on Gaeumannomyces graminis var. tritici and development of root-rot

This study provides evidence that egg-parasitic nematophagous fungi, Pochonia chlamydosporia, Pochonia rubescens and Lecanicillium lecanii, can also reduce root colonisation and root damage by a fungal pathogen. Interactions of nematophagous fungi with the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt), and their influence on severity of the root disease it causes were studied in laboratory and pot experiments. In Petri dish experiments the three nematophagous fungi reduced colonisation of barley roots by Ggt and also reduced necrotic symptoms. On the contrary, root colonisation by nematophagous fungi was unaffected by Ggt. In growth tube experiments, the three nematophagous fungi again reduced Ggt root colonisation and increased effective root length of barley seedlings. This was true for both simultaneous and sequential inoculation of nematophagous fungi versus Ggt. In the pot experiments the inoculum of the tested fungi in soil was applied in the same pot, as a mixture or in layers, or in coupled pots used for wheat grown with a split-root system. The nematophagous fungi P. chlamydosporia (isolate 4624) and L. lecanii (isolate 4629), mixed with Ggt or in split root systems with the pathogen, promoted growth of wheat (i.e. increased shoot weight), although no disease reduction was found. In split root systems, lower levels of peroxidase activity were found in seedlings inoculated with Ggt in combination with the nematophagous isolates 4624 and 4629 than when the take-all fungus was applied alone.Our results show that nematophagous fungi reduce root colonisation by Ggt, root damage and stress induced senescence in Ggt-inoculated plants.     

Hyphal density as a measure of suppression of Gaeumannomyces graminis var. tritici on wheat roots

Runner hyphae of Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici Walker on seminal roots of wheat seedlings were photographed and their length measured. As well, their length was estimated using the line-intercept method. The correlation of 0.904 between measured and estimated lengths of hyphae was highly significant. This line intercept method was used to estimate the density (length/unit area) of hyphae on roots of plants growing in the presence and absence of a soil suppressive to G. graminis var. tritici. Estimations were made eight times during 28 days growth at 15°C. In fumigated soil (non-suppressive) inoculated with 0.1% ground oat grain infested with G. graminis var. tritici, the density of hyphae on roots started to increase at five days compared with 15 days when soil there was a 10.8% cover of the root surface after 15 days when the hyphae had reached maximum density. Suppression to G. graminis var. tritici is normally detected by a difference in disease rating of roots at 28 days but this study has shown that suppression can be demonstrated by the difference in the density of hyphae if roots are examined between seven and 19 days.     

Cross-protection against the wheat and oat take-all fungi by Gaeumannomyces graminis var. graminis

Glasshouse experiments have shown that the prior colonisation of wheat roots by Gaeumannomyces graminis var. graminis, a fungus closely related to the wheat and oat take-all fungi but non-pathogenic to temperate cereals, reduced take-all infection along the roots. Cross-protected wheat plants produced grain yields significantly greater than those of unprotected plants but not significantly different to those of healthy wheat plants. A Phialophora-like fungus from grass roots did not confer the same degree of protection. There is some evidence that the cross-protection mechanism may be a specific host response nduced by var. graminis. The possible use of var. graminis in the biological control of take-all is discussed.     

Wheat-rhizoplane pseudomonads as antagonists of Gaeumannomyces graminis

The role of rhizoplane-inhabiting Pseudomonas spp as inhibitors of take-all on wheat was investigated. Apparent numbers of pseudomonads in wheat rhizoplanes and numbers that were antagonistic in vitro toward Gaeumannomyces graminis var, tritici did not differ when wheat was supplied with NH⁺₄-N or NO^?₃-N. More intense antagonism was expressed by colonies selected from soil treated with NH⁺₄-N than with NO^?₃-N, and from isolation media prepared at pH 5.5 rather than at 7.0. Antagonists were not recovered from methyl bromide-treated soil. Highly antagonistic pseudomonads were recovered from a wheat-monoculture soil which is considered suppressive toward the pathogen in the field, and were not recovered from a “nonsuppressive” soil. Pseudomonad antagonism ratings were inversely correlated with take-all severity in the suppressive soil, but not in the nonsuppressive soil. Pseudomonads were considered to be antagonists of G. graminis on rhizoplanes of wheat in a soil exhibiting the “take-all decline” phenomenon, but the significance of this interaction remains to be determined.     

Microbiology of roots infected with the take-all fungus (Gaeumannomyces graminis var. tritici) in phased sequences of winter wheat

Bacteria with possible relevance to the growth of the take-all fungus were counted from surfaces of lesioned and healthy roots of wheat growing in soil from a field monoculture system. Numbers showed short-term seasonal and long-term monocultural changes, which seemed to be genuinely associated with the monoculture. Bacteria were more numerous on lesioned than healthy roots. Only bacteria inhibitory to growth of Gaeumannomyces graminis on agar and Pseudomonas spp showed consistent changes irrespective of the source of the roots. Relationships were considered between the microflora on lesioned tissue and (a) severity of disease on roots supplying the lesions, and (b) infection produced on axenic seedlings inoculated with the lesioned tissue. Only total bacterial counts on the lesions from tillering and mature plants were positively correlated with disease on the donor roots. Only inhibitory bacteria on lesions from tillering and mature plants were positively correlated with disease on test seedlings. Pseudomonas spp showed no correlations. Interpretation of data differed with age of plant and the sequence in the monoculture from which plants or soil came.     

Age of wheat and barley roots and infection by gaeumannomyces graminis var tritici

Seminal roots of wheat and barley seedlings were inoculated with G. graminis var tritici on regions 0, 5- and 15-days old, and assessed for intensity and extent of infection after standard times. Wheat roots were most heavily infected on young regions, whereas barley roots were most heavily infected on old ones. The effect of root age in wheat was similar in both unsterile and aseptic conditions, so it could not be ascribed to saprophytic rhizosphere micro-organisms interacting with G. graminis.The contrasting results for wheat and barley are explained by a single hypothesis, based on decreasing host-resistance in the root cortex but increasing resistance at or near the endodermis as the roots age. It is suggested that, under some conditions, even small amounts of non-pathogenic root cortex death can enhance infection by G. graiminis. This interpretation may explain several aspects of take-all and its biological control by other dark mycelial parasites.     

Dynamics of root colonization by the take-all fungus, Gaeumannomyces graminis

The progressive colonization of wheat seminal roots by Gaeumannomyces graminis var. tritici was monitored following inoculation by single inoculum units. G. graminis grew equally well above and below inoculation sites prior to blockage of the stele but after this growth was favoured up roots, above inoculation sites, rather than down, resulting in an asymmetrical pattern of root colonization. This asymmetrical pattern was common to superficial and cortical runner hyphae. It is suggested that cessation of host assimilate supply to the distal portion of infected roots inhibited further extensive growth of G. graminis. This hypothesis was tested by comparing extents of colonization by G. graminis on seminal roots of wheat with normal, enhanced and diminished assimilate supplies. A diminished assimilate supply to infected roots retarded the extent of pathogen colonization.     

Pathozones of genetic subtypes of Gaeumannomyces graminis in cereals

The extent of damage to the host plant caused by Gaeumannomyces graminis var. tritici (Ggt) and var. graminis (Ggg) is a result of a net effect of host susceptibility and mycelium infectivity. The disease severity on cereal roots caused by G. graminis (Gg) fungi varies considerably depending on the genetic subtypes. Results of our rhizobox placement experiments additionally showed a subtype-specific effect of the spatial distance between host and fungus on the infection. The highest pathogenicity of each subtype was found in different zones of the root system: pathozones of different subtypes alternated along the root. The extent of the pathozone profiles did not depend on the infectivity of the inoculum and plant age. However, disease severity was shown to be affected by defence reactions of the host plant. An attack of a fungal subtype that is easily recognized by the host plant leads to defence reactions like increased root growth, thus minimizing the damage to the shoot. Detailed analysis showed that a Ggt subtype had a high potential for colonizing root laterals. It formed concentric zones of high colonization efficiency at a distance of ca. 5 cm around the shoot.     

Antagonists of Gaeumannomyces graminis from the rhizoplane of wheat in soils fertilized with ammonium- or nitrate-nitrogen

Take-all of wheat caused by Gaeumannomyces graminis var. tritici was less when soils in glasshouse pots were fertilized with NH₄⁺-N than with NO₃^?-N. The form of N did not alter countable populations of microorganisms in the rhizosphere or rhizoplane, but altered the numbers of bacteria and streptomycetes that inhibited the pathogen's growth in vitro. The pH of the medium used to isolate these microorganisms, whether similar or dissimilar to the pH of the rhizosphere, had some influence both upon countable populations and upon the proportions of antagonists. Highest counts of the rhizoplane microflora were on agar media with a pH similar to that of the soil. Most antagonists were isolated from a soil that is physically and chemically conducive to parasitism of wheat roots by Gaeumannomyces, but which contains a microflora suppressive toward the parasitic colonization of the roots. Isolates of the general bacterial flora, of Pseudomonas spp. and of streptomycetes, but not of Bacillus spp. inhibited the in vitro growth of G. graminis.     

Soils suppressive to Gaeumannomyces graminis var. tritici: Induction by other fungi

Rhizosphere soils were obtained from wheat plants growing in fumigated soil inoculated with one of the pathogens, Rhizoctonia solani, Gibberella zeae, Fusarium culmorum, Cochliobolus sativus and Pythium irregulare or one of the non-pathogenic fungi, Gaeumannomyces graminis var. graminis (Ggg) or a Phialophora-like fungus (Plf). Using a pot bioassay, these soils were tested for suppression of Gaeumanomyces graminis var. tritici (Ggt) and the fungus involved in the initial induction. G. zeae was the only fungus that induced suppression to Ggt and to itself. Ggg but not Plf induced suppression to Ggt although both fungi induced suppression to themselves.Fungi capable of inducing suppression of take-all have two characteristics in common, they induce suppression of themselves and their saprophytic survival is restricted to organic matter.     

The role of bacteria in the biological control of Gaeumannomyces graminis by suppressive soils

The suppression of Gaeumannomyces graminis var. tritici by certain soils or following certain soil treatments is considered to be an expression of either specific or general antagonism sensu Gerlagh (1968). Specific antagonism is effective in dilutions as high as 1 in 1,000, can be transferred from soil to soil, operates near or on wheat roots, is destroyed by 60°C moist heat for 30 min. or desiccation, is fostered by wheat monoculture but may be lost from a soil by fallow or rotation with certain crops, especially legume hay or pasture crops. Strains of Pseudomonas fluorescens may be involved. General antagonism is a soil property which cannot be transferred and is resistant to 80°C moist heat for 30 min, to methyl bromide and chloropicrin, but not to autoclaving. Take-all control by organic amendments, minimum tillage, or a soil temperature of 28°C may be expressions of increased general antagonism.In much of the southern Australian wheat belt, where take-all can cause heavy crop losses, some general but rarely specific antagonism is apparently operative. Both types of antagonism are probably operative in long-term wheat growing areas of the Pacific Northwest U.S.A. where take-all is virtually nonexistent.     

Soils suppressive to Gaeumannomyces graminis var. tritici: Effects on other fungi

The effect of soils suppressive to Gaumannomyces graminis var. tritici (Ggt) on the severity of root and crown rots caused by Rhizoctonia solani, Gibberella zeae, Pythium irregulare, Cochliobolus sativus and Fusarium culmorum was tested in pot bioassays. An induced suppressive soil was obtained from the rhizosphere of wheat plants grown at 15°C for 28 days in fumigated soil inoculated with live inoculum (colonized oat grain) of Ggt.Root rot caused by R. solani was significantly less in soil amended with either induced or naturally suppressive soil. Disease caused by the other pathogens was also reduced by the induced suppressive soil, with the least reduction occurring with F. culmorum.Colonization of the surfaces of seminal roots of wheat plants by Gaeumannomyces graminis var. graminis (Ggg) and a Phialophora-like fungus (Plf 119) was also studied using the line-intercept method. In non-suppressive soil the maximum area of the primary seminal root colonized by Ggg was 7.4 per cent and by Plf 119 was 3.3 per cent. Colonization of roots by Ggg and Plf 119 was reduced substantially by the addition of induced suppressive soil.     

Soil moisture affects the interaction between Gaeumannomyces graminis var. Tritici and antagonistic bacteria

Reduction in the soil moisture from a potential of ?10 to?108 kPa reduced the growth of Gaeumannomyces graminis. The antagonist Bacillus cereus only spread through the soil or sand at potentials between ?10 and ?60 kPa. The spread of B. pumilus was not affected by the water potential within the range tested. Both B. cereus and B. pumilus were less antagonistic in the drier sand and soil. When the bacteria and the fungus were grown together the maximum growth of the fungus occurred at those potentials when bacterial antagonism was reduced but the fungus was still able to grow.     

Growth of Gaeumannomyces graminis var. tritici in soil: Effects of temperature and water potential

The effects of temperature and water on the growth of the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt), were examined in two factorial experiments. The first examined the effects of temperature and water potential on the growth of two isolates of Ggt on agar media, using osmotically-adjusted water potentials. The second experiment was concerned with the growth of the Ggt isolates in one sterile and two natural soils at two water regimes in the absence of a living host. Three temperatures (10, 18 and 26°C) were used in these experiments. A third experiment determined growth through soil.Growth was greatest at high temperatures and low water potential in axenic culture, but in unsterile soil growth at different temperatures and water potentials was strongly influenced by competition from the soil biota. The best temperature for growth in unsterile soil was 18°C. Growth at 26°C in unsterile soil was greatly reduced, this being attributed to more intense microbial competition. In sterile soil Ggt grew equally well at 18 and 26°C. At 10°C, both isolates of Ggt grew better in unsterile soil than in sterile soil.Under suitable conditions Ggt grew out readily from infected straw into unsterile soil (up to 5 cm in 10 days) in the absence of a host plant, forming melanized, hyaline and branched hyphae. These hyphae were infectious after dry storage for 5 months in the laboratory. Ggt thus appears to be a more successful soil inhabitant than is widely believed. Our experiments could explain many of the host-based concepts related to field expression of disease.The technique presented here could be of value for testing the suppressiveness or conduciveness of soils by measuring fungal growth in soil.     

Infectivity of lesioned wheat root tissue related to the presence of dehydrogenase enzymes in hyphae of Gaeumannomyces graminis var. Tritici in the lesions

Transverse sections of lesioned tissue taken from wheat roots grown in soil naturally infested with Gaeumannomyces graminis var, Tritici were stained with trypan blue and the area of stele occupied by hyphae or by brown host deposits was measured. The area of mycelium in lesioned pieces taken from seedling or tillering plants and used as inoculum in host infectivity tests was positively correlated with the disease produced and the area of brown deposits in lesioned pieces taken from tillering or mature plants was negatively correlated. Whole pieces of lesioned tissue were examined cytochemically for glutamic and succinic dehydrogenases in the invading hyphae. Groups of host cells in the endodermal region were filled with hyphae showing positive reactions for both dehydrogenases (active) and separated by areas of brown discoloured host tissue containing few active hyphae. Less than half the discoloured lesion was occupied by active hyphae. The area of lesion containing hyphae with dehydrogenase enzymes was positively correlated with the measure of disease severity of the roots and with the infectiveness of the lesioned tissue when inoculated on to axenic wheat seedlings. The progress of infection in axenie seedlings inoculated at 3 or 8 cm from the seed differed with the two placements, notably in the host response and the growth of active hyphae in the cortex.     

Behaviour of cercosporella herpotrichoides and Ophiobolus graminis on buried wheat plant tissues

The survival of Ophiobolus graminis (Sacc.) Sacc. in buried wheat straw, but not of Cercosporella herpotrichoides Fron, was prolonged when nitrogen was added to soil. Five isolates of C. herpotrichoides survived similarly despite differences in their abilities to decompose cellulose and wheat straw. However, survival of O. graminis, at different temperatures, was entirely consistent with its cellulolytic activity. Unlike O. graminis, C. herpotrichoides survived up to 19 weeks in buried agar discs. These differences suggest contrasting means of survival of the pathogens.Both fungi showed short-term saprophytic activity in soil, growing into autoclaved wheat straws or coleoptiles only during the first few days after burial. An apparently increased competitive saprophytic colonization of straws at 10 than at 21°C, however, was largely an artefact of the “Cambridge Method” and not an indication of increased competitive saprophytism. Burial in unsterilized soil reduced the decomposition-rate of straws colonized by Chaetomium globosum Kunze and increased that of straws colonized by C. herpotrichoides. It also caused O. graminis to form lysis-resistant hyphae from previously unadapted ones.     

Bacterial colonization of seminal wheat roots

Changes in root morphology during root growth and concurrent bacterial colonization of the root surface were studied. Wheat seedlings were grown in soil so that their roots grew along a removable transparent plate on which root growth was recorded daily. The root surface microflora of 10mm lengths of root that had grown over 24 h, was counted on agar-plates and by fluorescence microscopy. Two maxima in bacterial colonization of the seminal axis of 10-day-old roots were demonstrated between root tip and root base. Fluorescence microscopy was also used to estimate at intervals along the root axis the proportions of lysing epidermis and cortex cells based on staining of nuclei with acridine-orange. Two regions of accelerated lysis of root tissue were demonstrated and are considered to be responsible for the two maxima in bacterial colonization of the root surface.