Effect of temperature on growth of some avirulent fungi and cross-protection against the wheat take-all fungus

The linear growth rates of Gaeumannomyces graminis var. graminis, G. graminis var. tritici, Phialophora radicicola var. graminicola and a lobed hyphopodiate Phialophora sp. were studied on agar at various temperatures between 5 and 30 °C and on wheat roots at two temperature regimes (12 h at 7°/12 h at 13 °C and 12 h at 17°/12 h at 23 °C). On agar at 30 °C, the isolates of G. graminis graminis grew faster than those of G. graminis tritici and Phialophora sp. but three isolates of G. g. graminis grew more slowly than the other two fungi at 5 and 10 °C. Two other isolates of G. g. graminis were cold-tolerant and had growth rates comparable to those of G. g. tritici and Phialophora sp. at 10 °C. The growth rates of Australian isolates of P. radicicola graminicolu were similar to that of a British isolate and were about a third to a half those of the other three fungi at most temperatures. The growth rates of the fungi on wheat roots at the low and high temperature regimes were correlated with the growth rates on agar at 10 and 20 °C respectively. The correlation was better at low temperatures r= 0.81) than at high temperatures (r = 0.62). Cross-protection experiments using two G. g. graminis isolates which grow poorly at temperatures below 15 °C and a cold-tolerant isolate each of G. g. graminis and Phialophora sp. showed that, while all four fungi protected wheat against take-all at high temperatures (17/23 °C) as evidenced by less severe disease and significantly greater dry weights, only the cold-tolerant fungi were effective at low temperatures (7/13 °C). The use of cold-tolerant isolates of avirulent fungi in field experiments may result in better protection in the early stages of wheat growth when Australian soil temperatures are mostly below 15 °C.     

Effect of osmotic potential on the growth of Gaeumannomyces graminis and Phialophora spp.

The linear growth of 10 isolates each of Gaeumannomyces graminis var. graminis, G. graminis var. tritici and Phialophora graminicola and five isolates each of G. graminis var. avenae and a lobed-hyphopodiate Phialophora sp. was studied on osmotically adjusted agar at 20 °C. While most isolates of G. graminis var. avenae ceased growing at osmotic potentials of -60 bars (1 bar = 10⁵ Pa), six out of 10 isolates of G. graminis var. tritici grew at that potential. The growth of all isolates of G. graminis var. tritici and var. avenae ceased at -70 bars. In contrast, four out of 10 isolates of P. graminicola grew at -70 bars, but all stopped growing at -80 bars. Most of the isolates of G. graminis var. graminis and the lobed-hyphopodiate Phialophora sp. grew at -70 bars while three out of 10 isolates of G. graminis var. graminis and one out of five isolates of the lobed-hyphopodiate Phialophora sp. were capable of growth at -80 bars. None of the fungi grew at -90 bars. Detailed studies of the growth of two or three isolates each of the five fungi at 10, 20, 30 and 35 °C were carried out on osmotic agar controlled by the addition of either sodium chloride or potassium chloride. In general, similar reductions in growth occurred with decreasing osmotic potential regardless of the solute used. At 10 and 20 °C., all three isolates of P. graminicola showed optimal growth at about -5 bars while the other fungi grew fastest at -1²middot; bars. At 30 °C., one isolate of the lobed hyphopodiate Phialophora sp. and two isolates each of P. graminicola, G. graminis var. tritici and G. graminis var. avenae grew optimally at osmotic potentials of -10 to -15 bars. The other isolate of the Phialophora sp. and two isolates of G. graminis var. graminis studied grew optimally at the highest potential (-1·2 bars). However, at 35 °C the last three fungi exhibited optimal growth at osmotic potentials of-10 to -20 bars. The ecological significance of these results is discussed in relation to cross-protection against the take-all fungi by the avirulent fungi.     

Saprophytic survival of Gaeumannomyces graminis and Phialophora spp. at various temperature-moisture regimes

The saprophytic survival of the pathogen, Gaeumannomyces graminis var. tritici and two isolates each of three avirulent fungi, G. graminis var. graminis, Phialophora graminicola and a lobed-hyphopodiate Phialophora sp. was studied in two soil types under controlled temperature and moisture conditions in the laboratory. In general, the fungi survived longest in the cool, dry soil (15°C, < -10 MPa) followed by the warm dry soil (30°C, < -10 MPa). All the fungi were virtually eliminated from the warm, moist soil (30°C, -0.3 MPa) after 3 months. Survival was intermediate under cool, moist conditions (15°C, -0.3 MPa). Under cool, moist conditions, G. graminis var. graminis survived better than the other three fungi in the first 3 months in both soil types and continued to do so for a further 3 months in one soil. Both isolates of the lobed-hyphopodiate Phialophora sp. survived poorly in the two soil types being almost eliminated after 3 months. There were considerable differences between the survival of the two isolates each of G. graminis var. graminis and P. graminicola, especially under cool, moist conditions. Of the six avirulent isolates studied, one isolate of G. graminis var. graminis (DAR24167) survived best under the three temperature-moisture regimes which showed differences. It also survived better than the take-all fungus under moist, cool conditions and at a comparable rate under dry conditions. Therefore, this variation in survival should be considered when selecting antagonists for the biological control of take-all.     

Methoxyhydroquinone, a Growth Inhibitor of Ophiobolus graminis, in Leaves of Oat Seedlings

A methanol extract of leaves of oat seedlings grown in sand cultures in the dark contained a compound which inhibited the growth of Ophiobolus graminis. The inhibitory factor was isolated and proved to be present in the plant as methoxyhydroquinone glucoside. The glucoside was readily hydrolysed to the corresponding aglucone. The methoxyhydroquinone, or possibly its oxydation product, methoxy-P-benzoquinone, was inhibitory to both Ophiobolus graminis var. graminis and Ophiobolus graminis var. avenae, whereas Fusarmm oxysporum var. lycopcrsici was not affected. Synthetic methoxyhydroquinone at 80 mg/l gave a 100% inhibition of Ophiobolus graminis var. graminis. After being exposed to 80 mg/l of the inhibitor for 24 h the mycelium was unable to initiate growth when transferred to a fresh nutrient solution. Only extracts from young leaves showed inhibitory activity, extracts from mature leaves giving no inhibition. The hydroquinone, or its glucoside, was not detected in roots of young seedlings, where avenacin was the only antifungal compound present.     

Pathogenicity of ‘take-all’ fungus to oats: Its relationship to the concentration and detoxification of the four avenacins

Data for inhibition of the growth of Gaeumannomyces graminis var. tritici (Ggt) and var. avenae (Gga), Phialophora radicicola and Fusarium avenaceum, caused by avenacins, are presented. The avenacins found in all oat species examined are sufficient in quantity to totally suppress growth of wheat ‘take-all’ (Ggt), even old roots containing 25 μg/g (fr. wt). Fungal variants that can also attack oats [var. avenae (Gga)] show considerable variations in their tolerance to avenacin A-1, ec₅₀ values being 5–80 μg/ml. Nevertheless, all Gga isolates maintained some growth at avenacin A-1 concentrations as high as 200 μg/ml and it is this ability to grow, albeit slowly, at high concentrations that is the critical difference between Gga and Ggt strains. The pathogenicity towards oats of a range of isolates of Gga is related to the fungicidal activity of avenacins. Gga pathogenicity is shown to increase with poor nutrition of the oat hosts (poor illumination, lack of minerals). Fungal detoxification of avenacins produces mono-deglucosylavenacin A-1, bis-deglucosylavenacin A-1 and, in one case, tris-deglycosylavenacin A-1. Ggt strains left avenacin A-1 almost unaffected giving only traces of mono-deglucosyl product. Gga strains bring about mono- and bis-deglucosylation whilst Fusarium avenaceum causes mainly bis-deglucosylation. Mono-deglucosylavenacin is shown to be less inhibitory to Gga than is avenacin A-1, whilst the bis-deglucosyl compound is still less inhibitory.     

Virus-like particles in the take-all fungus, Gaeumannomyces graminis

Isometric virus-like particles (VLP) measuring 35 nm and 27 nm occurred in cultured mycelium of Gaeumannomyces graminis var. tritici and G. graminis var. avenae. These VLP had, respectively, sedimentation coefficients (s°_{20, W}) 148S and 110S and ultraviolet absorption (maximum 260 nm, minimum 240 nm) typical of nucleoprotein (A260:280 = 1.6, A260:240 = 1.2). Preparations of the 35 nm particles had two major and one minor component in caesium chloride, and 27 nm particles had two components (buoyant densities 1.37, 1.36, 1.30, 1.35, and 1.29 g/cm³ respectively). Preparations of the 35 nm particles or 35 nm plus 27 nm particles had one major protein species with estimated molecular weight 70000 daltons. The 35 nm VLP were absent from 11 isolates of G. graminis var. tritici from first cereal crops after fallow or non-susceptible break crops; two of these contained the 27 nm particles. More than half of 145 isolates, from cereals after 2–12 consecutive susceptible crops, contained either 35 nm or 27 nm VLP. VLP were not confined to G. graminis isolates from soils exhibiting ‘take-all decline’ nor consistently associated with weak pathogenicity or with isolates of unusual growth, morphology, pigmentation, lysis or readiness to form perithecia. Isolates with one kind of particle were mostly more pathogenic and those with both kinds less pathogenic than isolates without VLP. The proportion of isolates with 27 nm and 35 nm particles increased progressively in samples from different consecutive crops during the first 9 years of cropping, then decreased. Isolates did not gain or lose VLP during infection and re-isolation from wheat seedlings grown in sand. Four ‘infected’ isolates were freed from VLP either by culturing ascospores or by growing hyphal tips excised from colonies kept near their thermal death point. Both VLP appeared in cultures which had undergone anastomosis with infected isolates.     

Detoxification of Benzoxazolinone Allelochemicals from Wheat by Gaeumannomyces graminis var. tritici, G. graminis var. graminis, G. graminis var. avenae, and Fusarium culmorum

The ability of phytopathogenic fungi to overcome the chemical defense barriers of their host plants is of great importance for fungal pathogenicity. We studied the role of cyclic hydroxamic acids and their related benzoxazolinones in plant interactions with pathogenic fungi. We identified species-dependent differences in the abilities of Gaeumannomyces graminis var. tritici, Gaeumannomyces graminis var. graminis, Gaeumannomyces graminis var. avenae, and Fusarium culmorum to detoxify these allelochemicals of gramineous plants. The G. graminis var. graminis isolate degraded benzoxazolin-2(3H)-one (BOA) and 6-methoxy-benzoxazolin-2(3H)-one (MBOA) more efficiently than did G. graminis var. tritici and G. graminis var. avenae. F. culmorum degraded BOA but not MBOA. N-(2-Hydroxyphenyl)-malonamic acid and N-(2-hydroxy-4-methoxyphenyl)-malonamic acid were the primary G. graminis var. graminis and G. graminis var. tritici metabolites of BOA and MBOA, respectively, as well as of the related cyclic hydroxamic acids. 2-Amino-3H-phenoxazin-3-one was identified as an additional G. graminis var. tritici metabolite of BOA. No metabolite accumulation was detected for G. graminis var. avenae and F. culmorum by high-pressure liquid chromatography. The mycelial growth of the pathogenic fungi was inhibited more by BOA and MBOA than by their related fungal metabolites. The tolerance of Gaeumannomyces spp. for benzoxazolinone compounds is correlated with their detoxification ability. The ability of Gaeumannomyces isolates to cause root rot symptoms in wheat (cultivars Rektor and Astron) parallels their potential to degrade wheat allelochemicals to nontoxic compounds.     

Immunoelectrophoretic characteristics ofOphiobolus graminis Sacc. as an aid in classification and determination

Antigens from four cultures ofO. graminis were compared immunoelectro-phoretically. Each culture produced a characteristic immunogram. More common antigens were found between the two cultures isolated from wheat or the two cultures isolated from oat than between a wheat and an oat isolate. Cell-wall antigens were the best reference antigens for serologic analysis of strain relationship. O. graminis antisera were cross-reacted with antigens from a number of other species of fungi. Relatively few of these cross-reacted with antisera to cell-wall antigens whereas more cross-reacted with antisera to whole-cell antigens.Immunoelectrophoretic analysis of antigens from a range of isolates ofO. graminis indicates specific immunograms which can be determined and separated from the immunograms developed by all other fungi when tested againstO. graminis antiserum. Immunoelectrophoresis can therefore be used as an aid in determiningO. graminis.     

Origin and Inheritance of Group I Introns in 26S rRNA Genes of Gaeumannomyces graminis

Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes. Received: 17 May 1996 / Accepted: 14 January 1997     

The Nature of the Resistance of Oats to the Take-all Fungus: II. INHIBITION OF GROWTH AND RESPIRATION OF OPHIOBOLUS GRAMINIS SACC. AND OTHER FUNGI BY A CONSTITUENT OF OAT SAP

Growth of Ophiobohu gramimt and of O. gramims var. avenae isinhibited by concentrations of 3·3-4·0µg./ml.,and respiration by concentrations of 55µg./nil. of a partiallypurified substance from oat-leaf sap. The two varieties appearto be equally sensitive. The filtrate of boiled sap is inhibitorybut here dilution of the sap permits better growth of isolatesof var. avenae. Sap from oat roots is inhibitory to O.graminisonly, and fractionation of the sap shows that the inhibitorcan be masked by a growth stimulant. Inhibition of growth andrespiration can be reduced by glutathione and ascorbic acid,particularly if the inhibitor and reducing agent are previouslyincubated together for a few hours, suggesting that the inhibitoris inactivated on reduction. The capacity of var. avenae toovercome inhibition in the favourable medium provided by thecrude sap more readily than can the type variety is suggestedas the cause of the slight differential activity of the filtrateof leaf sap and the full differential activity of the root sap.Susceptibility of oats to var. avenae would thus be due to conditionsenabling the fungus to overcome toxicity rather than to an absenceof toxicity. Activity of the inhibitor against growth and respiration ofa number of fungi and a few other organisms has been tested.Bacteria and oat and barley roots are not affected but abouthalf of the fungi tested are inhibited although none is as sensitiveas O. gramims. No members of the fungi imperfecti tested aresensitive.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism 3. Effect on Uptake of Potassium, Magnesium and Inorganic Phosphate

A venacin, the resistance factor in oat roots against Ophiobolus graminis var. graminis, and a related triterpeneglycoside, aescin, inhibited the uptake of K⁺ and Mg²⁺ in the fungal mycelium both in phosphate and succinate buffers. The uptake of the cations in Neurospora crassa was similarly inhibited when the inhibitors were dissolved in phosphate or acetatebuffer, while no decrease in the uptake of K⁺ and Mg²⁺ was observed when the inhibitors were dissolved in succinate buffer. The uptake of cations in Aspergillus niger and Pythium irregulare was more or less unaffected by aescin. The uptake of inorganic phosphate was in no case inhibited, but some decrease of the accumulation of inorganic phosphate in Ophiobolus graminis and Ncurospora crassa due to inhibitor treatment in phosphate buffer was observed. No accumulation of Ca²⁺ was observed in any of the tested fungi.     

Interactions between Sarocladium oryzae and stem attacking fungal pathogens of rice

In laboratory tests Sarocladium oryzae, the sheath rot pathogen of rice was found to inhibit the mycelial growth of other stem-attacking rice pathogens. Among those inhibited, Sclerotium oryzae and Gaeumannomyces graminis var. graminis were most sensitive while Pyricularia oryzae and Rhizoctonia solani were less sensitive. Tissue-based tests made with rice culm segments established that Sarocladium oryzae inhibits mycelial growth and delays sclerotium formation in R. solani. Cerulenin, the toxin produced by Sarocladium oryzae showed a toxicity pattern towards rice pathogens similar to that of Sarocladium oryzae. The stem rot pathogen, Sclerotium oryzae was most sensitive to cerulenin. In two greenhouse experiments, IR58 rice plants inoculated with Sarocladium oryzae alone or together with Sclerotium oryzae, G. graminis var. graminis or R. solani were found to have reduced plant height and increased tiller number. Sheath rot severity increased when Sarocladium oryzae was inoculated as a single pathogen or together with others. Sheath rot inoculation reduced stem rot in rice plants by 76 and 58%, respectively, in Experiment 1 and 2. By its known antagonistic interaction towards stem rot and crown sheath rot pathogens which are sensitive to it and by other unknown interactions, sheath rot emerges as the dominant disease.     

Variability among strains of Trichoderma harzianum in their ability to reduce take-all and to produce pyrones

Antagonism tests on agar-plates and glasshouse screening indicated that three isolates of Trichoderma harzianum varied in their ability to antagonize the take-all fungus (Gaeumannomyces graminis var. tritici). Isolate 71 which was the most effective in suppressing take-all of wheat, produced two pyrones and other undetermined analogues. Isolates of T. koningii and T. hamatum shown to suppress take-all, produced a simple pyrone compound. Although T. harzianum isolates 70 and 73 did not produce any pyrones, they reduced the disease albeit to a much lesser extent than isolate 71; with isolate 73 showing distinct host growth promotion effects. It is proposed that the success of isolate 71 of T. harzianum was related to the pyrones it produces and that the ability of isolates 70 and 73 to reduce take-all may be related to mechanisms other than those involving antibiotics.     

Disease in wheat genotypes naturally infected with Gaeumannomyces graminis var. tritici

Wheat genotypes consisting of seven homozygous lines and 40 segregate families were studied at two sites naturally infested with the take-all pathogen, Gaeumannomyces graminis var. tritici. The numbers of seminal and coronal roots infected with G. graminis and other root pathogens were recorded. The genotypes differed in infection with G. graminis, with little evidence of genotype × environment interactions. The incidence of G. graminis and Rhizoctonia solani was negatively associated, but the association did not greatly influence differences between wheats in infection with G. graminis. The distribution of R. solani was negatively associated with the severity of take-all at only one site. Of symptoms of infection with G. graminis, wheat genotypes differed most in incidence of deadheads, but differences were not consistent over environments, and were associated with earliness of maturity. Wheats differed more in expression of disease than in infection with G. graminis. The course of disease was deduced from associations between the incidence of pathogens and components of plant growth and yield. G. graminis was the dominant pathogen at both sites, and caused a yield loss of 0–15% at one site, and an average 62% loss at the other. More components of yield were affected where disease was most severe.     

Infection process of Gaeumannomyces graminis var. graminis on the roots and culms of rice

Crown sheath rot, caused by the ascomycete Gaeumannomyces graminis var. graminis that infects the root and the base of the culm of rice, causes early grains maturation, tiller death and reduced yield. As a paucity of information exists in the literature on the rice‐G. graminis var. graminis interaction at the microscopic level, this study aimed to gain novel insights into the infection process of this pathogen in the root and culm of rice using both light and scanning electron microscopy. In the roots, the fungus initially colonized the epidermal, exodermal and sclerenchyma cells. At 15 days after inoculation (dai), fungal hyphae colonized the cortex and clusters of perithecia were observed in the roots. At 20 dai, the fungus reached the central cylinder, and an intense fungal colonization at the base of the culm was observed that resulted in the formation of a mycelial mat on both adaxial and abaxial surfaces of the leaf sheaths. At 25 dai, fungal growth was noticed in the parenchyma cells, vascular bundles and airspaces. Perithecia emerged through the base of prophyllum and from the first leaf sheath at 30 dai. The results of this study provide new insights into the infection process of G. graminis var. graminis in rice and may help to find better control measures in reducing crown sheath rot development.     

Control of Ophiobolus patch in Agrostis turf using avirulent fungi and take-all suppressive soils in pot experiments

The incorporation of avirulent fungi such as Gaeumannomyces graminis var. graminis, an avirulent isolate of G. graminis var. tritici, a Phialophora sp. with lobed hyphopodia synonymous with Phialophora radiciola var. radicicola sensu Deacon and P. radicicola var. graminicola at the time of seeding Agrostis turf in pots of sterilised soil completely controlled Ophiobolus patch disease. The addition of a 5 mm layer of take-all suppressive (TAS) soils, artifically developed by the repeated addition of live mycelium of the varieties avenae, tritici and graminis of G. graminis to soil, controlled the disease to a lesser extent. However, a 20 mm layer of a TAS soil developed from live mycelium of G. g. avenae almost completely suppressed the disease. A survey of 66 golf and bowling greens in four states of Australia showed that P. r. graminicola was the most prevalent avirulent fungus.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

A venacin, the resistance factor in oat roots against Ophio-bolus graminis var. graminis, and a related triterpeneglycoside, aescin, induced a rapid release of K⁺ from mycelia of Opbio-bolus graminis and Neurospora crassa, suspended in phosphate buffer. N. crassa also released Mg²⁺ whereas no outflux of Mg²⁺ was found from O. graminis. The inhibitors induced a release of inorganic phosphate into acetate buffer from Neurospora crassa. The amount of inorganic phosphate in the mycelia decreased when O. graminis and N. crassa were treated with the inhibitors in phosphate buffer. In other media the inhibitors had weak or no effects on the ion contents of the mycelia. The effect of aescin was low in Aspergillus niger and nil in Pythium irregulare. However, high amounts of K⁺, Mg²⁺, and phosphate ions were lost to the medium when the mycelium of P. irregulare, washed with distilled water, was suspended in different buffers. The ions lost were reabsorbed during the experimental period. The leakage of ions indicates that the plasma membrane of the growth sensitive fungi is severely affected by the inhibitors, while a corresponding effect on the growth insensitive fungi does not take place.     

Biofumigation: Isothiocyanates released frombrassica roots inhibit growth of the take-all fungus

The presence of root tissue of the brassicas canola and Indian mustard inhibited growth of pure cultures of the fungal pathogen which causes take-all of wheat [Gaeumannomyces graminis (Sacc.) Arx and Oliver var.tritici, abbreviated as Ggt]. Ggt growth was generally inhibited more in the presence of Indian mustard roots than canola roots. Dried irradiated roots were consistently effective in reducing Ggt growth, but growth inhibition by young live roots and macerated roots was not consistent. The inhibitory compound(s) were shown to be volatile because the symmetry of Ggt growth was not affected by the proximity of theBrassica tissue. Volatile breakdown products from maceratedBrassica roots were identified using a gas chromatograph-mass spectrometer. The major compounds found were isothiocyanates (ITCs). Canola roots released mostly methyl ITC and Indian mustard roots released mostly phenylethyl ITC. Low concentrations of these and related compounds inhibited growth of Ggt in pure culture when supplied as the vapour of pure chemicals in concentrations within the range expected during breakdown ofBrassica roots in soil.     

An Enzymic Basis for Pathogenic Specificity in Ophiobolus graminis

Avenacin, the glucosidic inhibitor present in oat roots, isacted on by a specific glucosidase produced by Ophiobolus-graminisvar. avenae, which destroys biological activity, as does hydrolysiswith 0.1 N. HCl. Neither O. graminis itself nor any other fungustested produces this enzyme. It is suggested that the resistanceof oats to O. graminis and its susceptibility to var. avenaedepend on an inhibitor-inactivating enzyme complex, and thisis compared with the antibiotic-inactivating enzyme complexfound in penicillin-producing moulds and resistant penicillinase-producingbacterial strains; such a complex may be concerned in othercases of pathogenic specificity. The way in which a varietyof O. graminis pathogenic to oats may have arisen is discussed.     

The Nature of the Resistance of Oats to the Take-all Fungus

The two varieties of the take-all fungus, Ophiobolus graminisand 0. graminis var. Avenae, show a differential reaction tosap extracted from oats; the type variety, which is incapableof causing a lasting infection of oats in vivo, is inhibitedby the sap in vitro, whereas var. Avenae, pathogenic to oats,can grow in the sap. The inhibitory action of the sap is notdue to a lack of food material required by the fungus, but toa specific substance or toxin. The method used for assayingtoxicity is described. The toxin is thermostable, moderately stable on storage, virtuallyinsoluble in non-polar solvents, and soluble in acetone, water,and methanol. Methanol extraction of the ether-washed residueof the filtrate from boiled sap from oat leaves results in asemi-purified substance which, when added to 2 per cent. Yeastrelsolution, reduces growth of 0. graminis to half that of thecontrol at a concentration of to 10 p.p.m. The inhibitor is produced in considerable quantity in the leavesand stems of oats and in smaller quantity in the roots. It appearsto accumulate mainly during the period of active growth andto be less active, or present in smaller concentration, in adultplants. It can be detected in the seminal roots throughout theirexistence, although during the first 3 weeks of growth 0. graminiscan invade the cells of the cortex. It is present in greaterquantity in the crown roots, which are never penetrated. A similar inhibitor of 0. graminis can be extracted from Arrhenatherumelatius, but not from other grasses which, both in the fieldand in pot experiments, appear to be equally resistant.