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.     

Factors impacting the activity of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens against take-all of wheat

Take-all, caused by Gaeumannomyces graminis var. tritici, is an important soilborne disease of wheat worldwide. Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are biocontrol agents of take-all and provide natural suppression of the disease during wheat monoculture known as take-all decline. To identify factors that could contribute to the effectiveness of 2,4-DAPG producers in take-all suppression, P. fluorescens strains Q8r1-96 (genotype D) and Q2-87V1 (genotype B; reduced antibiotic production) were tested against three pathogen isolates differing in sensitivity to 2,4-DAPG (LD5, ARS-A1 and R3-111a-1) and two wheat cultivars (Tara and Buchanan). The antibiotic sensitivity of the pathogen and cultivar significantly affected the level of take-all suppression by Q8r1-96 and Q2-87V1; suppression was greatest with LD5 and Tara. Q8r1-96 suppressed ARS-A1 and R3-111a-1 on Tara but not Buchanan, and Q2-87V1 failed to suppress either pathogen isolate on either cultivar. Q8r1-96 colonized the rhizosphere of Tara and Buchanan grown in soil similarly, but 2,4-DAPG accumulation was higher on the roots of Buchanan than Tara. 2,4-DAPG at 7.5 μg mL⁻¹ reduced the growth of roots of both cultivars, and 10 μg mL⁻¹ caused brown necrosis and tissue collapse of seedling roots and reduced root hair development. The half-life of 2,4-DAPG in the rhizosphere was estimated to be 0.25 days. These results suggest that several interconnected factors including sensitivity of G. graminis var. tritici to 2,4-DAPG, wheat cultivar, fluctuations in populations of 2,4-DAPG producers, and antibiotics accumulation in the rhizosphere will impact the robustness of take-all suppression by P. fluorescens in the field.     

A new growth medium for the study of siderophore-mediated interactions

Summary A microbial growth medium, RSM, was developed to study the role of siderophores (microbial Fe-transport compounds) in the inhibition of the take-all pathogen, Gaeumannomyces graminis var. tritici, by Pseudomonas putida strain B10. The inorganic constituents of the medium were designed to mimic the rhizosphere while the organic composition was designed to promote rapid growth and siderophore production. The antibiosis experiments were highly reproducible and the antagonism appeared to be due to production of pseudobactin, the siderophore of B10. On plates amended with chrome azurol S, G. graminis did not produce siderophores while other fungi did. The growth of G. graminis on plates prepared with Fe chelate buffers was inhibited at a free ferric ion concentration of 10^–24.6 M, although three other fungi were not inhibited, even at 10^–25.5 M, presumably due to their greater production of siderophores. In liquid medium amended with Fe chelate buffers, both the doubling time and the lag phase of P. putida increased as the free ferric ion concentration was reduced. A wide variety of fungi and bacteria were found to grow on this medium. Because the inorganic composition of RSM is based on that of the rhizosphere, the development of this medium may be a first step towards the study of the chemistry and biology of the rhizosphere under well defined conditions.     

Mycorrhizal fungi increase biocontrol potential of Pseudomonas fluorescens

Wheat roots are susceptible to colonisation by soil-borne pathogens, such as Gaeumannomyces graminis var. tritici (Ggt), which causes the globally important disease take-all, and mutualistic arbuscular mycorrhizal fungi (AMF). Certain rhizosphere fluorescent Pseudomonas strains have received much attention as potential biocontrol agents given their ability to produce antibiotics, such as 2,4-diacetylphloroglucinol (DAPG), that confer a measure of plant protection. Here we show that Pseudomonas fluorescens only produced DAPG in the presence of soluble carbon from soil containing either Ggt or AMF, and production increased by two orders of magnitude in response to both AMF and Ggt. Encouragement of mycorrhizal colonisation may therefore offer a sustainable strategy for protection against take-all.     

Studies on soil fumigation—II: Effects on bacteria

In a South Australian wheat-field soil the viable counts of “total” aerobic bacteria and of fluorescent pseudomonads were initially greatly depressed by fumigation with 220 kg·ha^?1 chloropicrin (CP) or with a combined application of 220 kg·ha^?1 of chloropicrin and 220 kg·ha^?1 methyl bromide (F). Bacterial numbers rose sharply within 10 days of the completion of fumigation. For a further 14 days the fluorescent pseudomonads formed the major part of the aerobic bacterial population counted and over 5 months later their numbers in F-treated soil remained about 10 times higher than in untreated soil. Numbers of aerobic spore-formers rose more slowly after CP or F treatment. but then remained significantly higher over the 159 days of the trial. Fumigation with 220 kg·ha^?1 of methyl bromide alone (MB) had little effect on soil bacterial numbers.A check of random isolates revealed a predominance of Gram-negative organisms in soil treated with CP, this dominance decreasing with time, whereas MB treatment did not result in any detectable change.Fluorescent pseudomonads from rhizospheres of wheat plants in soil fumigated with CP contained smaller proportions of strains antagonistic in vitro to Gaeumannomyces graminis var. tritici than isolates from MB-treated soil or from untreated soil.     

Manganese-oxidizing ability of isolates of the take-all fungus is correlated with virulence

Two successive experiments described here show that the Mn-oxidizing ability of isolates of the take-all fungus Gaeumannomyces graminis var. tritici is positively correlated with the isolate virulence. Isolates of G. graminis var. tritici were screened for virulence on wheat in soil culture pot tests and visually scored for Mn-oxidizing capacity on agar plates. A comparison between experiments showed that the Mn-oxidizing ability of an isolate may be attenuated, as is virulence, with subculturing. Using a solution culture system, we calculated the rate of oxidation of Mn²⁺ by a virulent isolate, Ggt 500, as 4 nmol g^-1 (dry weight hyphae) h^-1. The significance of these results to the epidemiology of the take-all disease of wheat is discussed.     

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.     

MICROBIAL POPULATIONS AND NITROGEN IN SOIL GROWING CONSECUTIVE CEREAL CROPS INFECTED WITH TAKE-ALL

Annually, for the past 12 years, a consecutive cereal-cropping sequence was begun. During 1969–72 soil was collected from plots with similar fertilizer treatments, but with different sequences of crops. In 1971 and 1972 wheat seedling bioassays showed that take-all disease became miximal after two or three crops, decreased to the fifth or sixth crop and then remained fairly constant in the‘take-all decline’state. The sequence with maximum disease had most NH₄⁺ -N and least NO₃^- -N in the rhizosphere soil in spring 1972, but there were no similar relationships in bulk soil in either of 2 years. Soil and rhizoplane populations of ammonifying and denitrifying bacteria were notably smaller in autumn 1970 and 1971 in short cereal sequences (2-5 yr.) than in the longer sequences. After γ-irradiating or autoclaving irradiated soils, all sequences contained much NH₄⁺ -N and although disease developing from added inoculum differed among soils, it did not follow the take-all decline pattern. Diffusate from sterilized soil favoured growth of the pathogen (Gaeumannomyces graminis var. tritici) least when it originated from the sequence that supported maximum disease.     

Ammonium and nitrate in the rhizosphere of spring barley, hordeum vulgare L., and take-all disease

The incidence and severity of take-all disease, due to Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici Walker, was observed on spring barley plants growing in soil in two glasshouse experiments. Soil amendments of NH⁺₄-N significantly increased the number of diseased plants and roots during the first month after germination in comparison with controls unamended with N (P < 0.05). No significant difference in the incidence of take-all disease was detected between more mature barley plants growing in soil amended with either NH⁺₄ or NO^?₃-N and unamended controls. The least take-all disease in 3 month-old barley plants was observed when N was supplied as foliar sprays of urea at 0.5 mg N kg^?1 soil (P < 0.01). There was no significant correlation between the degree of infection and the NH⁺₄-N to NO^?₃-N ratio in the rhizosphere soil     

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.     

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.     

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.     

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.     

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 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.     

Infection of wheat seminal roots by varieties of Phialophora radicicola and Gaeumannomyces graminis

The growth of isolates of Phialophora radicicola var. radicicola, P. radicicola var. graminicola, Gaeumannomyces graminis var. graminis, G. graminis var. tritici and Leptosphaeria narmari was compared on the coleoptiles and roots of wheat seedlings. Fungal growth was measured as the extent and density of dark runner hyphae. All except P. radicicola var. graminicola grew on coleoptiles and all grew on roots although only G. graminis var. tritici extensively colonized the root stele. Growth rate on roots was positively correlated with that on agar, P. radicicola var. graminicola and L. narmari growing at about half the rate of the other fungi; hyphal density was high for P. radicicola var. graminicola but relatively low for the other fungi. For P. radicicola var. radicicola, P. radicicola var. graminicola and G. graminis var. tritici growing from buried inocula, the extent and density of hyphae up roots towards the seed was similar to that down, but G. graminis var. tritici caused chocolate-brown stelar discoloration up roots only.Root invasion by P. radicicola var. radicicola, P. radicicola var. graminicola and G. graminis var. tritici was described from sections. Each gave a different pattern of hyphae and host response within an inoculum layer, and progressive changes occurred away from the inoculum. Studies of the rate of penetration by each fungus and the rate and pattern of death of cortical cells explained the differences between fungi. G. graminis var. tritici penetrated living cells in advance of other soil micro-organisms, and hence by hyaline hyphae inducing much lignituber formation as a host resistance reaction. P. radicicola var. graminicola penetrated only senescent or dead cells in association with other soil microorganisms, and hence by dark hyphae, inducing little lignituber formation. P. radicicola var. radicicola was intermediate in all these respects. The high hyphal density of P. radicicola var. graminicola was due to the colonization of cortical cells and spaces by dark, clearly visible, rather than hyaline hyphae, which are invisible in unstained roots. Cell death in the outer cortex explained the observed progressive restriction of growth by all fungi to the inner cortex with increasing distance from the inoculum. Spread by G. graminis var. tritici up roots was ectotrophic relative to the stele but down roots hyphae spread rapidly within the stele. Stelar reactions suggested as resistance mechanisms occurred up roots only. Their absence down roots is attributed to infection disrupting stelar transport.     

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.     

稻秸杆覆盖对小麦根围拮抗细菌组成和小麦纹枯病发生的影响

To assess the effect of rice straw mulching on changes of antagonistic bacteria and the incidence of wheat sharp eyespot, a multi-year field study was performed to compare unmulched plots and the plots mulched with rice straw for two or three years. Bacterial and fungal populations were evaluated in the cultures prepared from the wheat rhizosphere and bulk soils. Rice straw mulching increased the number of pseudomonas colony forming units in wheat rhizosphere and bulk soils. The proportion of total bacteria that were fluorescent pseudomonads was higher in mulched than in unmulched soil. Bacterial isolates antagonistic to Rhizoctonia cerealis were identified using an inhibition zone test. A series of these isolates were typed by partial sequencing of the 16S rRNA gene. Pseudomonads had higher antagonistic activity against R. cerealis than other species, and more than 80% of rhizosphere fluorescent pseudomonads were antagonistic to R. cerealis. The disease indices were lower in the mulched plots than in the unmulched control. These results suggest that rice straw mulching in a rice-wheat rotation increases the number of fluorescent pseudomonads. Additionally, these fluorescent pseudomonads may contribute to the control of wheat sharp eyespot.     

Mycophagous soil amoeba: Interactions with three plant pathogenic fungi

An amoeba of the order Leptomyxida was isolated from wheat take-all decline soil and was found to attack and lyse hyphae and spores of Gaeumannomyces graminis var. tritici and Phytophthora cinnamomi. The amoeba enveloped portions of hyphae of both fungi and penetrated the cell walls by means of fine holes. One-week old chlamydospores and hyphal swellings of P. cinnamomi were also attacked in this way, protoplast lysis being completed within 1 h. Hyphal fragments which could be ingested by the amoeba were lysed leaving amorphous cell debris. Three-week old chlamydospores of P. cinnamomi were enclosed within large food vacuoles and completely digested in about 20 h. Pigmented conidia of Cochliobolus sativus were transported across the substratum for up to several hours but were not perforated or lysed.     

EFFECT OF CHLORIDE FERTILIZERS AND LIME ON WHEAT GRAIN YIELD AND TAKE-ALL DISEASE

One experiment was initiated in the fall of 1991 to evaluate the effect of chloride (Cl) fertilizers on the suppression of take-all disease (Gaeumannomyces graminis var. tritici Walker) in winter wheat (Triticum aestivum L.). Preplant and topdress rates of potassium chloride (KCl) and calcium chloride (CaCl₂) (0, 34, 67 and 101 kg Cl ha^?1) were applied each year. In 1995, plots were split in half whereby one half received 2.24 Mg of 76% ECCE lime ha^?1 to elevate the pH and potentially increase disease incidence. Wheat grain yield was not affected by lime applications in any year (1995–1999). Plots exhibited visual symptoms of take-all in almost all years, however, grain yields increased in only two of eight years by the application of CaCl₂ and KCl. Applied fertilizer Cl for take-all disease suppression was inconsistent, even following the application of lime where increased soil pH can increase disease severity.