Arbuscular mycorrhizal fungal-induced alteration to root architecture in strawberry and induced resistance to the root pathogenPhytophthora fragariae

Three strawberry cultivars Elsanta, Cambridge Favourite and Rhapsody were inoculated with eitherGlomus fasciculatum orGlomus etunicatum and their growth compared with non-inoculated plants. The roots of all inoculated plants were 55 to 70% colonised after 98 days. Increases in both root and shoot dry weights were measured. Root architecture was also determined and increases in branching were evident in AMF colonised root systems. The remaining plants were then inoculated with the root pathogenPhytophthora fragariae and allowed to grow for a further 58 days before harvest. In two of the cultivars, Cambridge Favourite and Elsanta, AMF reduced root necrosis by approximately 60 and 30% respectively. Only in the least susceptible cultivar, Rhapsody, was no reduction measured in AMF colonised plants. There were differences in the control conferred by the two arbuscular mycorrhizal fungi and this suggests there may be practical benefits of inoculation. Relationships between the presence of roots of different orders, on inoculation with the pathogen, and subsequent necrosis provided a mechanism for identifying root-architecture driven alteration to susceptibility. Root system necrosis was positively correlated with the proportion of the root system made up of higher order roots (3° to 4°) in non-colonised plants and negatively correlated in AMF colonised plants. These data suggest that root-architecture changes are not important per se but factors expressed concurrently may be.     

Initial incidence and development of red core of strawberry

The initial incidence of the disease caused by Phytophthora fragariae in healthy stock of strawberry, cv. Merton Princess, was not affected by different systems of cultivation or by no cultivation for several months before preparing infested soil for planting. In disease development studies, healthy plants which were planted in infested soil in April and sampled at 35-day intervals showed symptoms of red core on 1 September. Disease severity (mean % number of diseased roots) increased with time and, when averaged over intervals of 70 days, the greatest increases occurred between the end of July and the beginning of October and between December and February. Whereas the former was due to infection of plants in which the total numbers of new roots were low, the latter was due to an increase in the numbers of diseased roots. Most primary roots were produced in October and early November and 75% of the final total were formed by 15 December. Healthy plants of the cvs Cambridge Favourite and Redgauntlet, dug from the field at monthly intervals and challenged with zoospores, were less susceptible in May and June than in other months. It is suggested that a fungicide applied in late September or October would protect the maximum number of new roots and achieve the optimum effect for a single application.     

Sensitivity of the ELISA Test to Detect Phytophthora fragariae var. rubi in Raspberry Roots

A commercial serological, multiwell assay kit was used to assess the detection limits of Phytophthora fragariae var. rubi in raspberry roots. Detection limits in time lapse after inoculation, were assessed after inoculation of root systems of raspberry plants by zoospores of P. fragariae var. rubi. In extracts taken 3-9 days after inoculation, the pathogen was detected from the fourth day after inoculation. In a test series of simulated P. fragariae var. rubi infection where 0.25, 0.5, 1.0 and 1.5% of infected root tissue respectively, were mixed with healthy tissue (w/w), it was possible to detect the pathogen at 0.25% of simulated infection level. The results obtained show the possibility of an early detection of small amounts of antigen by the ELISA test procedure used. This enhance possibilities for early diagnosis and thereby more effective prevention of Phytophthora diseases in raspberry.     

Detection of cross-reactive antigens shared byFusarium oxysporum andGlycine max by indirect ELISA and their cellular location in root tissues

Pathogenicity test ofFusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium ofF. oxysporum. Polyclonal antisera were raised against the mycelial suspension ofF. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.     

Growth promotion and disease suppression ability of Pseudomonas fluorescens in acid lime

Biochemical analysis was carried out for 10 isolates of Pseudomonas fluorescens. All isolates were found positive for siderophore and indole acetic acid production (except Pf IX) and phosphate solubilisation (except Pf VII). Biochemically efficient strains (Pf I, Pf IV, Pf VII and Pf IX) were selected for management of root rot, collar rot and damping off caused by Phytophthora parasitica, Pythium sp. and Fusarium solani. The activity of defence-related enzymes like esterase, peroxidase and polyphenol oxidase was also detected by polyacrylamide gel electrophoresis (PAGE). Consistent appearance of esterase isozyme bands was visualised, in the range of 0.193–0.349. The highest Rm value 0.349 was observed on Pf IV. Whereas, for peroxidase, Rm value ranged between 0.302 and 0.373 and for polyphenol oxidase, it was in the range of 0.211–0.800. P. fluorescens Pf IV was found significantly effective to arrest the per cent mycelial growth of P. parasitica (55.20%), Pythium sp. (65.33%) and F. solani (64.67%). Among bioagents, seed treatment and soil drenching with Pf IV at 15 and 30 days after sowing were found effective to reduce per cent disease incidence (30.55%) at 120 days after emergence. Seed treatment with copper oxychloride at 3g/kg seed and metalaxyl at 2 g/kg seed were also found effective.     

Evaluation of antiserum raised againstPestalotiopsis theœ for the detection of grey blight of tea by ELISA

Polyclonal antiserum was raised against the mycelial extract ofPestalotiopsis theœ and immunoglobulin fractions were purified by ammonium sulfate fractionation and chromatography on DEAE-Sephadex. In enzyme-linked immunosorbent assay, antiserum dilution up to 1∶16000 detected homologous antigen at a 5 mg/L concentration, and at 1∶125 antiserum dilution fungal antigens could be detected at a concentration as low as 25 μg/L. In fifteen varieties of tea tested, originating from Darjeeling, UPASI and Tocklai breeding stations, absorbance values of infected leaf extracts were significantly higher than those of healthy extracts at a concentration of 40 mg/L in indirect ELISA. ELISA-positive material was detected in tea leaves as early as 12 h after inoculation withP. theœ. At antiserum dilutions up to 1∶125, the pathogen could be detected in inoculated leaf extracts up to antigen concentration of 2 mg/L. The antiserum reacted with two other isolates ofP. theœ tested but not with the antigens from mycelial extracts ofGlomerella cingulata andCorticium invisum or with extracts of tea leaves inoculated with these pathogens. The results demonstrate that ELISA can be used for early detection ofP. theœ in leaf tissues even at a very low level of infection.     

Interaction of Pratylenchus penetrans and Rhizoctonia fragariae in Strawberry Black Root Rot

A split-root technique was used to examine the interaction between Pratylenchus penetrans and the cortical root-rotting pathogen Rhizoctonia fragariae in strawberry black root rot. Plants inoculated with both pathogens on the same half of a split-root crown had greater levels of root rot than plants inoculated separately or with either pathogen alone. Isolation of R. fragariae from field-grown roots differed with root type and time of sampling. Fungal infection of structural roots was low until fruiting, whereas perennial root colonization was high. Isolation of R. fragariae from feeder roots was variable, but was greater from feeder roots on perennial than from structural roots. Isolation of the fungus was greater from structural roots with nematode lesions than from non-symptomatic roots. Rhizoctonia fragariae was a common resident on the sloughed cortex of healthy perennial roots. From this source, the fungus may infect additional roots. The direct effects of lesion nematode feeding and movement are cortical cell damage and death. Indirect effects include discoloration of the endodermis and early polyderm formation. Perhaps weakened or dying cells caused directly or indirectly by P. penetrans are more susceptible to R. fragariae, leading to increased disease.     

Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f. sp. ciceri in chickpea

Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. A marked increase in shoot and root length was observed in P. fluorescens treated plants. The isolates of P. fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium. oxysporum f.sp. ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control. Varied degree of protection against Fusarium wilt was recorded with chemical inducers. The reduction in disease was more pronounced when chemical inducers were applied with P. fluorescens. Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers. A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested. Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application. All the isolates of P. fluorescens produced SA in synthetic medium and in root tissues. HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates. 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog. After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.     

Genomic-assisted characterisation of Pseudomonas sp. strain Pf4, a potential biocontrol agent in hydroponics

In an attempt to select potential biocontrol agents against Pythium spp. and Rhizoctonia spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus Enterobacter, whereas nine strains belonged to the genus Pseudomonas. In in vitro assays, one strain of Pseudomonas sp., Pf4, closely related to Pseudomonas protegens (formerly Pseudomonas fluorescens), showed noteworthy antagonistic activity against two strains of Pythium aphanidermatum and two strains of Rhizoctonia solani AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for in vivo treatments on lamb’s lettuce against R. solani root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 10⁵?CFU?g^?1 of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that Pseudomonas sp. Pf4 has the potential to produce an arsenal of secondary metabolites (plt, phl, ofa and fit-rzx gene clusters) very similar to that of the well-known biocontrol P. protegens strain Pf-5.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Mycosphaerella pinodes in pea seeds

A polyclonal antiserum was raised against soluble mycelial extracts of Mycosphaerella pinodes aiming at pathogen detection in infected pea seeds by ELISA. When tested against the homologous antigen, it allowed the detection of 5 ng fungal soluble protein ml^-1 buffer, by double-antibody sandwich ELISA (DAS-ELISA). Positive reactions were obtained with isolates of M. pinodes of wide geographical origins but also with all tested isolates of Ascochyta pisi and Phoma medicaginis var. pinodella, two closely related pathogens forming with the target organism the Ascochyta complex. Out of the 11 other genera of pea seed-borne fungi tested, only two (Alternaria sp. and Stemphylium sp.) cross-reacted strongly by both antigen-coated plate (ACP-ELISA) and DAS-ELISA. Cross-absorption of the crude antiserum could not lead to a species-specific antiserum; however, a combination of P. medicaginis var. pinodella and Stemphylium sp. antigens resulted in an antiserum preferentially recognising A. pisi and M. pinodes. The cross-absorbed antiserum detected 50 and 500 ng of fungal protein ml^-1 buffer and healthy seed extracts respectively. DAS-ELISA proved suitable for the detection and quantification of M. pinodes in infected pea seeds tested singly.     

Nachweis von Phytophthora fragariae Hickman in Wurzeln der Erdbeerkultursorte 'Tenira' mit Hilfe des enzyme-linked immunosorbent assay (ELISA)

Detection of Phytophthora fragariae Hickman in roots of strawberry cultivar ‘Tenira’ by enzyme-linked immunosorbent assay (ELISA) Phytophthora fragariae Hickman is detected by ELISA in roots of strawberry cv. ‘Tenira’. Because of the high sensitivity of ELISA presence on fungal antigen was demonstrated before symptoms are detected in microscopical investigations.     

Fungi isolated from Sclerotia ofSclerotium cepivorum and from soil and their effects upon the pathogen

Summary Data are presented on the antagonistic effects of the fungi isolated from sclerotia ofSclerotium cepivorum and from nonrhizosphere soil taken from around the roots of infected onions upon mycelial growth and sclerotial germination ofS. cepivorum. Most of the isolated fungi especiallyPenicillium species were antagonistic to mycelial growth. Sclerotial germination was slightly inhibited by diffusates of these fungal isolates. Testing the antifungal effect of someAllium extracts against the fungal isolates by the inhibition zone method showed that garlic extract has the greatest antifungal effects and onion extract is the least potent. However, spore germination tests indicated that onion extract completely inhibits the spore germination of all test fungi. The role of host-plant extracts in stimulating sclerotial germination is discussed.     

Variation in the virulence of Verticillium dahliae from strawberry

Isolates of V. dahliae from strawberry, tested in 1968 against cv. Cambridge Vigour, showed differences in virulence; in cvs Talisman and Cambridge Favourite there were no significant differences between plants that were inoculated and those that were not. Evidence from this experiment, and from an earlier non-replicated experiment with Cambridge Favourite, in which differences of virulence had been apparent, showed that environmental factors can cause variations in disease development that are of similar magnitude to those attributable to genetic variations in host and parasite.     

Armillaria jezoensis,a new symbiont ofGaleola septentrionalis (Orchidaceae) in Hokkaido

NineArmillaria isolates obtained from the roots ofGaleola septentrionalis in Hokkaido were identified asA. jezoensis by means of mating tests. Cultures of these isolates were similar in colony morphology, mycelial growth and rhizomorph formation on each of malt extract-dextrose agar (MDA), potato-dextrose agar (PDA), andG. septentrionalis root extractdextrose agar (GDA) media, showing better mycelial growth and rhizomorph formation on GDA medium.     

Comparison of Pratylenchus penetrans Infection and Maladera castanea Feeding on Strawberry Root Rot

The interaction of lesion nematodes, black root rot disease caused by Rhizoctonia fragariae, and root damage caused by feeding of the scarab larva, Maladera castanea, was determined in greenhouse studies. Averaged over all experiments after 12 weeks, root weight was reduced 13% by R. fragariae and 20% by M. castanea. The percentage of the root system affected by root rot was increased by inoculation with either R. fragariae (35% more disease) or P. penetrans (50% more disease) but was unaffected by M. castanea. Rhizoctonia fragariae was isolated from 9.2% of the root segments from plants not inoculated with R. fragariae. The percentage of R. fragariae-infected root segments was increased 3.6-fold by inoculation with R. fragariae on rye seeds. The presence of P. penetrans also increased R. fragariae root infection. The type of injury to root systems was important in determining whether roots were invaded by R. fragariae and increased the severity of black root rot. Pratylenchus penetrans increased R. fragariae infection and the severity of black root rot. Traumatic cutting action by Asiatic garden beetle did not increase root infection or root disease by R. fragariae. Both insects and diseases need to be managed to extend the productive life of perennial strawberry plantings.     

Effects of Pratylenchus penetrans on development of strawberry wilt caused by Verticillium dahliae

The nematode Pratylenchus penetrans or the closely related P. fallax occurred in three out of four strawberry plantations infested with Verticillium dahliae surveyed in Kent. When plants of cv. Cambridge Vigour were inoculated with P. penetrans and V. dahliae together in field ‘micro-plots’ the nematode increased the rate of wilt development except when the concentration of V. dahliae microsclerotia in the soil was very low. In a pot experiment, under conditions not conducive to symptom expression, the incidence of infection was increased by nematodes in Cambridge Favourite, Cambridge Vigour and Redgauntlet; the incidence was least in Red-gauntlet, the most resistant cultivar. The nematode appeared to cause local changes in the root cortex which aided hyphal penetration of the adjacent tissues. Growth of strawberry plants in the absence of V. dahliae was not significantly affected even by large populations of the nematode, but growth was reduced by V. dahliae at all rates of inoculation in the field. Infection by Verticillium did not affect the total numbers of nematode per plant at the end of the experiments, although the number per g fresh weight was greater on the small amount of white root on severely diseased plants than on the larger amount on healthy or mildly diseased plants.     

Biocontrol of root-knot nematode Meloidogyne incognita by the isolates of Pseudomonas on tomato

Biocontrol of root-knot nematode Meloidogyne incognita was studied on tomato using 15 isolates of fluorescent Pseudomonads isolated from pathogen suppressive soils. Pseudomonas aeruginosa (isolates Pa8, Pa9 and Pa3) caused greater inhibitory effect on hatching of M. incognita than other isolates. In addition, isolates Pa8, Pa9 and Pa3 caused greater colonisation of tomato roots and also caused a greater increase in the growth of tomato seedlings. These isolates also caused a greater increase in growth of tomato and higher reduction in galling and nematode multiplication in a green house test than is caused by other isolates. Isolates Pf1, Pf5, Pf6 and Pa13 were unable to increase growth of tomato and caused less reduction in galling and nematode multiplication compared to other isolates. Only 10 isolates produced siderophores on chromo-azurol sulfonate (CAS) agar medium and isolate Pa12 showed greater production of siderophore followed by Pa11, Pa9, Pf10, Pa3 and Pf5. Similarly, isolates Pa14, Pa12, Pf10, Pa9, Pa8, Pa7 and Pa6 produced greater amount of HCN than the other isolates tested. Isolates Pa8 and Pa9 showed greater production of IAA than the other 13 isolates tested. This study suggests that P. aeruginosa isolates Pa8 and Pa9 may be used for the biocontrol of M. incognita on tomato.     

A rapid serological test for detecting Fusarium oxysporum f.sp. narcissi in Narcissus

Polyclonal antiserum was elicited against a strain of Fusarium oxysporum f.sp. narcissi (GCRI80/26) and a specific and sensitive enzyme-linked immunosorbent assay developed. Antiserum raised to cell wall fractions gave better recognition than that to cytoplasmic fractions. Recognition was equally good in artificially and naturally infected bulbs. Little cross-reactivity in bulb tissue was shown by three other bulb-rotting fungi. Nine isolates of F. oxysporum f.sp. narcissi from a wide geographic area gave similar results in an indirect ELISA of mycelial extracts, although some cross-reactivity was observed with two other Fusarium spp. Four Fusarium spp. and four other fungi showed little cross-reactivity. Ten days after inoculation the pathogen was readily detected in the base plate area of three Narcissus cultivars and points remote from the inoculation site in the most susceptible cultivar. A direct correlation was observed between positive results in the enzyme-linked immunosorbent assay and recovery of the pathogen on selective medium.     

Oxidising activity in root extracts from plants inoculated with virus or buffer that interferes with ELISA when using the substrate 3,3', 5,5'-tetramethylbenzidine

In indirect ELISA using protein A-horseradish peroxidase (HRP) as enzyme conjugate and 3,3′, 5,5′-tetramethylbenzidine (TMB) as substrate, extracts of roots of all cucumber, Chenopodium quinoa and Petunia hybrida plants previously inoculated with virus or buffer produced A₄₅₀ values up to seven-fold greater than those for comparable shoots or for extracts of roots from undisturbed, uninoculated plants, irrespective of the virus antiserum used for detection. This effect was also produced in tests in which no HRP conjugate was used, indicating that root extracts from virus-infected or physically injured plants, but not healthy uninjured plants, contain high levels of a factor able to oxidise TMB. The HRP conjugate/TMB substrate version of ELISA is therefore not reliable for detecting viruses in root extracts of herbaceous plants. In contrast, non-specific reactions were not obtained with root extracts, and viruses were reliably detected, when protein A-alkaline phosphatase was used as conjugate and p-nitrophenyl phosphate as substrate.     

Glucose, and not sucrose, is transported from wheat to wheat powdery mildew

The main host carbon energy source transferred from wheat leaves (Triticum aestivum L.) to wheat powdery mildew (Erysiphe graminis f.sp. tritici) has been investigated in three ways. When the uptake of sugars by isolated mycelial suspensions was examined, the uptake rate for glucose was considerably higher than that for a range of other solutes. Analysis by high-performance liquid chromatography of leaf and mycelial extracts following uptake of sugars into infected leaf pieces confirmed that sucrose was rapidly hydrolyzed in the leaf; no sucrose or fructose could be detected in mycelial extracts. Furthermore, studies of the uptake of asymmetrically labelled sucrose indicated that this sugar is cleaved prior to uptake by the pathogen. Thus several lines of evidence show that glucose, and not sucrose, is the major carbon energy source transferred from host to fungal mycelium. Received: 11 November 1998 / Accepted: 18 January 1999