Fungal Assciations: II. Cultural Studies on Rhizoctonia solani Kuhn, Fusariutn solani Snyder and Hansen, and Other Fungi, and Their Interactions

Several types of interaction between Rhizoctonia solani, Fusariumsolani, and Phoma foveata were found when these fungi were grownon potato-dextrose agar. After being used by Rhizoctonia a potatomash medium gave better growth of Rizoctonia and Fusarium thanit did when the medium was initially used by Fusarium; and thiswas so whether the reaction of the spent medium was readjustedor not. It is suggested that potato mash medium used by Fusariumcontains a thermostable factor(s) affecting the subsequent growthof Rhizoctonia or Fusarium. The range of pH values suitable for Rhizoctonia growth was narrowerthan that for Fusarium, optimum values being approximately 5•9for the former and 7•8 for the latter. In mixed culturesof the two fungi on potato-dextrose agar adjusted to differentpH values, the fungus for which the reaction of the medium wasmore suitable usually became visually predominant after sometime. A study of various carbon sources showed that poor growth ofRhizoctonia was obtained when pectin was used as the sole sourceof carbon. On a pectin-agar medium, the rate of growth of aRhizoctonia colony increased on the sector which lay towardsan adjacent Fusarium colony; also, after the two fungi camein contact, there was more rapid growth of Rhizoctonia roundthe Fusarium colony than elsewhere. On a synthetic liquid mediumwith pectin as the carbon source better Rhizoctonia growth wasobtained when Fusarium-spent medium was added to it than whenRMzoctoma-spent medium was added. Rhizoctonia showed partial deficiencies in thiamine, biotin,and inositol. Both the extract of Fusarium mycelium, grown onvitamin-free medium, and the Fusarium-spent medium, stimulatedthe growth of Rhizoctonia on vitamin-free medium.     

Fungal Associations: III. The Role of Pectic Enzymes on the Synergistic Relation between Rhizoctonia solani Kuhn and Fusarium solani Snyder and Hansen, in the Rotting of Potato Tubers

The greatest activity of protopectinase obtained from the growthof Rhizoctonia solani and Fusarium solani on autoclaved potatoplugs occurred at pH 6.5, and greatest activity of the ‘lossof viscosity’ enzyme was found at 6–5 for Rhizoctonia,and between 6.5 and 8.3 for Fusarium. Protopectinase enzymeobtained from double infections of the Fusarium spp. with Rhizoctonia,or by mixing the enzymes of individual Fusarium spp. with Rhizoctoniaenzyme, were more active than the enzymes from single inoculations.Cylindrocarpon radicicola enzyme was more active when obtainedfrom a pure culture than from double infection. Similarly, mixingthis enzyme with the enzyme of Rhizoctonia reduced its activity.The evidence indicated that the protopectinase of Rhizoctoniawas similar to that of Cylindrocarpon and differed from thatof the Fusarium spp. Using paper partition chromatography, two bands from Rhizoctoniacrude enzyme had a stimulatory effect on Fusarium enzyme, whileonly one band from Fusarium enzyme stimulated Rhizoctonia enzyme. The purified enzyme of Rhizoctonia degraded pectin to galacturonicacid. Fusarium pure enzyme degraded pectin to an intermediatestage. A mixture of the two enzymes degraded pectin to galacturonicacid, without the intermediate stage formed by Fusarium alonebeing detected. The role played by pectic enzymes upon the synergistic relationof Rhizoctonia solani and Fusarium solani on rotting potatotubers is discussed.     

Control of Fusarium solani rot of potato tubers with fungicides

Tecnazene (up to 33 ppm) and dichloran (up to 500 ppm) had little effect on germination of spores or growth of Fusarium solani isolated from and causing a rot of potato tubers; they also did not decrease rotting when applied to wounds later inoculated with the pathogen. Benomyl and thiabendazole (up to 500 ppm) also had little effect on spore germination but did greatly decrease growth at 5 ppm. A pronounced pink coloration developed in cultures growing slowly in the presence of benomyl; a similar though less striking effect appeared in agar cultures containing thiabendazole. Benomyl suspended in water or diluted with Fuller's earth gave good control of rotting when applied to wounds inoculated later with F. solani. Still better control was obtained with thiabendazole; dusts containing 1% a.i. substantially decreased rots and those containing 10 % a.i. gave almost complete control when applied to wounds shortly before inoculation. Thiabendazole was also very effective when used 24 h after inoculation and a fair measure of control was obtained when it was applied 24 h later. Benomyl and thiabendazole placed on apparently intact surfaces of tubers caused tissue 5 mm deep to become toxic to F. solani 10 days later, and, unexpectedly, this tissue prevented spore germination. Fuller's earth alone substantially decreased rotting. The results obtained suggest that dusts containing thiabendazole have some promise for the control of Fusarium rots of potato tubers, especially of early crops.     

Stem canker (Rhizoctonia solani) of maincrop potatoes.

In two years, potato plants were sampled at 1- or 2- weekly intervals from plots planted with seed tubers bearing sclerotia of Rhizoctonia solani (black scurf) and with seed without sclerotia either infested or not with cultures of R. solani at planting. Sprouted King Edward seed was used in 1981 and sprouted and non-sprouted King Edward and Pentland Crown seed in 1982. In both years 60–80% of shoots from seed with sclerotia and 90% of shoots from seed inoculated at planting were affected with stem canker. Most disease developed before shoots emerged although it gradually increased later when new shoots arising both from seed tubers or as branches on shoots with damaged apices (pruned shoots) became infected before they emerged. Sprouting seed tubers bearing sclerotia decreased the disease on both cultivars but with soil-applied inoculum the disease was more severe on plants from sprouted than non-sprouted seed. Some stolons were infected by R. solani soon after they developed and incidence of infection later increased. Thirty to 50% of stolons were infected on plants from infected seed tubers and 60% on plants with soil-applied inoculum. With both cultivars and sources of inoculum about 70% of the infected stolons had their apices killed (pruned).     

NITROGEN METABOLISM IN WHITE POTATO TUBER TISSUE INFECTED WITH FUNGI. CHANGES IN THE ALCOHOL-SOLUBLE AND -INSOLUBLE NITROGEN FRACTIONS

An increase of the ratio insoluble N/soluble N in potato tubertissue (var. Bintje) infected with Gibberella saubinetii orCerato-cystis fimbriata and incubated at 25° could onlybe demonstrated in samples cut from the area invaded by thefungi. In the non-infected controls some increase was foundin tissue close to the cut surface due to wound-reaction. The fungi hardly penetrated tuber tissue of var. Koopmans Blauwe,while tubers of var. Alpha were rather susceptible. Tissue ofvar. Bintje turned out to be rather resistant. Incubation at30° or 33° resulted in a more limited penetration bythe fungi. No relation exists between the value of the ratioinsoluble N/soluble N or the protein content of the host tissue,and the grade of resistance to the infection. Again, an increaseof insoluble N could only be observed in mycelium-containingsamples. The mycelia of both fungi had a ratio insoluble N/soluble Nwhich was much higher than the ratio of normal potato tissue.It was possible to explain the increase of the ratio in invadedtissue on the basis of the presence of hyphal substances. No evidence was obtained for a synthesis by the host tissueof insoluble nitrogenous compounds prior to, or concomitantwith, the increase of respiration rate after infection. To whatextent a wound-reaction played a role in the infected tuberparts is uncertain. The possibility of a simultaneous synthesisand breakdown of proteins after infection without a rise ininsoluble nitrogen is discussed. (Received February 3, 1966; )     

Seasonal Changes in Molecular Forms of Acid Phosphatase and Ribonuclease of Potato Tubers and the Effects of Infection by Phytophthora erythroseptica

Sequential changes in total activity and molecular forms ofacid phosphatase and ribonuclease from potato tubers were studiedby seasonal assays and Sephadex gel filtration. Ribonucleaseand p-nitrophenyl phosphatase activity fluctuated during storageof tubers, while ß-glycerophosphatase declined toa low level coincident with initiation of sprout growth. Inrecently-lifted tubers acid phosphatase activity occurred ina single high molecular weight peak. Two new forms of lowermolecular weight appeared during ageing of stored tubers. Theinfluence of infection by a tuber-rotting fungus, Phytophthoraerythroseptica,on these seasonal changes was variable. No consistent effectson total hydrolase activities were observed, while post-infectionalchanges in molecular forms included a pronounced shift in themajor acid phosphatase peak. The possible significance of thismolecular weight change in infected samples is discussed inthe light of recent evidence concerning the sub-unit structureof acid phosphatase from potato tubers.     

Comparison of temperature effects on the in vitro growth and disease development in potato tubers inoculated with bacteria Pectobacterium atrosepticum,P carotovorum subsp. carotovorum and Dickeya solani

The relationship between the rate of in vitro growth of bacterial isolates of Pectobacterium atrosepticum, P. carotovorum subsp. carotovorum and Dickeya solani and their pathogenicity was investigated in tubers of two potato cultivars at four temperatures ranging from 18°C to 30°C. The rate of in vitro growth was highly positively correlated with the number of rotted tubers (r ranged from 0.91 to 0.93) and with the weight of macerated potato tissue, which, however, was only found for P. carotovorum and D. solani (r = 0.76; r = 0.91, respectively) and not for P. atrosepticum. The weight of macerated tissue increased with the temperature, but significant differences between species of bacteria were observed only at 26°C and above, at which temperatures D. solani was the most aggressive, followed by P. carotovorum and P. atrosepticum. Almost all potato tubers inoculated with bacteria showed symptoms of soft rot at 26°C and 30°C, but the number of rotting tubers at lower temperatures (22°C and 18°C) decreased significantly. The lowest disease incidence, 11% of tubers with symptoms, was observed for the D. solani and cultivar Sonda at 18°C, what was also confirmed in a separate experiment with tubers from four potato cultivars inoculated with the highly aggressive isolate of D. solani. At temperatures from 18°C to 30°C, the differences in disease severity between potato cultivars with various resistance to bacteria increased in line with temperature, while the differences in disease incidence decreased.     

Transmission of Fusarium solani var. coeruleum and F. sulphureum from seed potatoes to progeny tubers in the field

In experiments in three years, seed tubers were inoculated before planting with either Fusarium solani var. coeruleum or F. sulphureum to initiate a rot, or were contaminated by dipping in soil slurries containing spore suspensions of one or other of the pathogens. Transmission to progeny tubers was tested by uniformly wounding and incubating tubers and by dilution plating of soil samples. In two years, transmission of F. sulphureum was greater from highly contaminated than from rotting seed and was greater on cv. Pentland Crown than on cvs Desiree and Maris Piper. F. solani var. coeruleum appeared to be transmitted most readily from rotting seed and Maris Piper was the cultivar most extensively contaminated. In experiments with different harvest dates, transmission of both fungi from highly contaminated seed could be detected by late June or mid-July. More progeny tuber wounds rotted in F. sulphureum than in F. solani var. coeruleum plots and in one year, F. sulphureum caused more rots on cv. Record than on cv. Maris Piper. These differences between the pathogens may be related to their differing abilities to sporulate underground on the surface of seed tubers and on stem bases.     

Biology,Epidemiology and Management of Rhizoctonia solani on Potato

Black scurf and stem canker on potato is an economically important disease complex, causing both quantitative and qualitative damage to potato crops which occurs in potato production areas throughout the world. The ribosomal DNA internal transcribed spacer sequence analysis is currently accepted and a commonly used method for classifying Rhizoctonia species and anastomosis groups (AGs). To date, 13 AGs have been recognized. The updated AG distribution in potato worldwide production areas confirm the status of AG‐3 as the most prevalent AG in potato and reflects the population dynamics of the pathogen probably due to global trading of tubers. As R. solani is a tuber‐ and soilborne pathogen, the ability to detect its levels in the seed tubers and in the soil and predict the potential damage is an important factor in controlling the disease. Effective disease management of Rhizoctonia disease requires implementation of an integrated disease management approach and knowledge of each of its stages. Although the most important control measures are cultural, chemical control (either by seed tuber‐ or in‐furrow treatments) is still an important tool in reducing the damages caused by R. solani.     

Detection of Helminthosporium solani from soil and plant tissue with species-specific PCR primers

Two PCR primer pairs specific for Helminthosporium solani, which causes silver scurf on potato tubers, were designed from nucleotide sequences of the nuclear ribosomal internal transcribed spacer regions of H. solani. Both primer pairs amplified a single product with DNA from 48 North American and European isolates of H. solani, but not with DNA from 42 other fungi. Primers also amplified a single product with DNA extracted from silver scurf lesions on potato tubers and other plant tissue inoculated with spores of H. solani. Detection of the fungus in infested soil was only possible with nested PCR and after processing soil with a bead beater. Specific amplification of H. solani DNA can be used to study the saprophytic and pathogenic activity of this fungus in soil and plant tissue.     

Development of potato blight Phytophthora infestans after planting infected seed tubers

About 1000 blight-infected seed potato tubers, usually of the cultivar King Edward, were planted for 9 yr and the subsequent plants examined until the disease had developed in the plots. Haulm infection originated each year from the seed tubers and occurred first on basal leaves. When tubers were inoculated with a complex race of P. infestans this race was recovered from the leaves and from the soil near the seed tuber. Transmission of infection from soil to leaves was demonstrated by splash of artificially contaminated soil to leaves suspended above the soil. In 4 yr, plants were grown on flat rows as well as on ridges. In 2 yr, when emergence was almost complete, infected stems were observed on otherwise normal plants. In the first year 0.6% grew on ridges and 3.0% on the flat and in the second all grew on the flat (5.3%). Only seven of the 43 plants had more than one infected stem. Flat plots had a significantly higher number of stemdplant than ridge plots, but this bore no relation to numbers of infected stems. When flat plots which had developed affected plants had soil replaced as ridge plots, no further infected stems were observed. Such stems continued to develop on flat plots. No prematurely dead stems were observed below soil level when all plants were dug. Underground portions of most infected stems showed little evidence of P. infestans which was found only at about soil level. Infection appeared to occur first in this area.     

Susceptibility of potato tubers to infection by Phytophthora infestans

Phytophthora infestans infects King Edward potato tubers more readily through inoculated eyes than through lenticels, but more lenticels than eyes became infected when whole tubers were sprayed with inoculum. The resistance of lenticels but not of eyes increased as tubers aged. The spores did not infect through intact periderm. The likelihood of tubers on plants grown in pots becoming infected by sporangial suspension poured on to the soil increased the nearer the tubers were to the soil surface, the stem, or the side of the pot. Naturally infected tubers, and those sprayed with sporangial suspension, had most eyes infected at the rose end, and most lenticels infected on the middle region of the tuber. Of naturally infected tubers, on which the site of infection could be identified, most were infected through eyes at the rose end.     

Studies on Endogenous Bacteria in Potato Tubers Infected by Phytophthora erythroseptica Pethybr

Infection of potato tubers by the soft-rotting fungi Phytophthoraerythroseptica and Pythium debaryanum resulted in multiplicationof endogenous tuber bacteria. This effect was not evident afterinfection by dryrotting fungi. Some of the bacteria isolatedproduced pectolytic and hemicellulolytic enzymes in vitro andwere themselves capable under certain conditions of degradingtuber tissue.     

THE INFECTION OF PLANTS BY VIRUSES THROUGH ROOTS

Roots of young tomato plants became infected when inoculated with tomato bushy stunt, tobacco mosaic, and potato X viruses. Root infections also occurred when these viruses were added to soil or culture solutions in which plants were growing.
The viruses were sometimes localized around their initial entry points in roots; sometimes they invaded the root system but not the shoots, and sometimes they produced full systemic infection of roots and shoots. In some experiments, but not all, systemic infections were more frequent when the upper tap root or superficial roots were inoculated than when fibrous roots were inoculated.
In both tomato and potato, virus X spread from diseased to healthy plants sharing the same culture solution, if their roots were in contact, but not otherwise. Infection of the roots of potato plants by inoculation, produced only one plant with virus-infected haulms, although several had infected tubers.     

Genetic modification of potato against microbial diseases: in vitro and in planta activity of a dermaseptin B1 derivative, MsrA2

Dermaseptin B1 is a potent cationic antimicrobial peptide found in skin secretions of the arboreal frog Phyllomedusa bicolor. A synthetic derivative of dermaseptin B1, MsrA2 (N-Met-dermaseptin B1), elicited strong antimicrobial activities against various phytopathogenic fungi and bacteria in vitro. To assess its potential for plant protection, MsrA2 was expressed at low levels (1–5 g/g of fresh tissue) in the transgenic potato (Solanum tuberosum L.) cv. Desiree. Stringent challenges of these transgenic potato plants with a variety of highly virulent fungal phytopathogens—Alternaria, Cercospora, Fusarium, Phytophthora, Pythium, Rhizoctonia and Verticillium species—and with the bacterial pathogen Erwinia carotovora demonstrated that the plants had an unusually broad-spectrum and powerful resistance to infection. MsrA2 profoundly protected both plants and tubers from diseases such as late blight, dry rot and pink rot and markedly extended the storage life of tubers. Due to these properties in planta, MsrA2 is proposed as an ideal antimicrobial peptide candidate to significantly increase resistance to phytopathogens and improve quality in a variety of crops worldwide with the potential to obviate fungicides and facilitate storage under difficult conditions.     

A comparison of Fusarium avenaceum and Fusarium caeruleum as causes of wastage in stored potato tubers

Fusarium avenaceum is reported for the first time as a cause of rotting of potato tubers in Britain. The progress of rotting in tubers infected with F. avenaceum has been compared with dry rot due to F. caeruleum in the laboratory, clamp and potato store. Of the four varieties, Majestic, King Edward, Doon Star and Arran Pilot, tested for susceptibility, King Edward was the most susceptible to F. avenaceum and Doon Star to F. caeruleum.
Optimum temperatures for growth on potato-dextrose agar were 20-25 C. for F. avenaceum and 20 C. for F. caeruleum ; maximum temperatures were > 30 and 30 C. respectively. For infection of wounded potato tubers, cardinal temperatures for F. avenaceum were similar to those on agar, but for F. caeruleum the optimum was 15 C. and the maximum 25 C. The optimum temperature for rotting tended, with both species, to be higher in the more susceptible potato varieties. At low temperatures F. caeruleum caused quicker rotting than did F. avenaceum , even though its rate of growth on agar was scarcely more than half that of the latter.
High humidity favoured rotting especially by F. avenaceum; F. caeruleum was more tolerant of relatively low humidity. Both species caused quicker rotting in the clamp than in store, even though there was no appreciable difference in mean temperature between the clamp and the store. This was attributed to the higher atmospheric humidity in the clamp.     

Effect of certain cultural practices on susceptibility of potato tubers to soft rot disease caused by Erwinia carotovora pv. carotovora

Erwinia soft rot causes destructive and serious damage to many vegetable crops including potato in the field, transit and storage periods. The effect of certain cultural practices on the susceptibility of potato tubers to soft rot bacteria was studied and the results of this work can be summarised in the following: potato tubers harvested on 1 May first exhibited the highest disease incidence compared with those harvested on 15 May or 30 May. Harvesting on 15 June resulted in the lowest disease infection. The application of high levels of nitrogen fertiliser as urea (46.5%), ammonium nitrate (31%) and ammonium sulphate (20.5%) resulted in an increase of the susceptibility of potato tubers to bacterial soft rot disease. In contrast, the addition of phosphorous as superphosphate (15.5%) fertiliser caused the reverse effect. The addition of potassium as potassium sulphate (48%) alone at any of the tested levels showed no effect. The susceptibility of potato tubers to bacterial soft rotting disease was increased by increasing storage periods at 4°C for 1, 2, 3 and 4 months. Spraying copper sulphate exhibited the highest decrease in soft rot incidence disease followed by manganese, zinc and iron. However, spraying of boron increased susceptibility to the disease. Potato tubers obtained from plants sprayed with copper and stored for different periods showed the lowest susceptibility to disease incidence. Tuber sprayed with zinc, iron, manganese and finally boron came next.     

Survival of the causal agents of 'early-dying disease' (Verticillium wilt) of potatoes

Although the isolation of Verticillium albo-atrum and V. dahliae from soil and dried moribund stems following infection of a potato crop proved extremely difficult, both fungi were equally capable of overwintering in these substrates and of inducing disease in a subsequently planted susceptible crop. In the absence of a susceptible crop some weed species became colonized. The two species, however, appeared to differ in their capacity for survival both beneath a monocotyledonous crop and within the potato tubers. Colonization of the roots of wheat, barley, oats, rye and maize was observed with V. dahliae but not with V. albo-atrum. The latter appeared to be capable of prolonged survival in the tubers, whereas V. dahliae did not remain viable in storage over winter. Consequently only tubers infected with V. albo-atrum produced infected plants. The presence of the fungi within the tubers affected neither dormancy nor the initial development of the sprouts. Some correlation was noted between tuber size, the percentage of tubers infected, the distribution of V. albo-atrum within the tubers and the development of disease in plants subsequently grown from these tubers.     

The infection of oranges by Trichoderma viride and mixed infection by Trichoderma viride and Penicillium digitatum

Trichoderma viride spores applied in water to apparently uninjured skin of oranges do not cause lesions. Adding orange juice, rind extract, citric acid or orange essential oil did not influence infection. Oranges became infected only when the stem-end cuts or wounds deeper than 6 mm into oil vesicles were inoculated. Sound oranges in contact with decayed oranges did not become infected. Diphenyl-impregnated wrappers reduced infection. A mixed inoculum of T. viride and Penicillium digitatum caused as fast rotting as P. digitatum, which caused faster rotting than T. viride alone. Lesions infected with P. digitatum could become infected by T. viride but those caused by T. viride did not become infected by P. digitatum. T. viride was antagonistic to P. digitatum in vivo and in vitro, possibly because it produces a heat-labile diffusible substance toxic to P. digitatum.     

Root rot disease incidence and severity on some legume species grown in Samsun and the fungi isolated from roots and soils

Abstract

Root rot disease is very common in the bean, soybean, faba bean and pea plants growing areas in Samsun province. Disease incidence and severity were detected the highest at 93.8% and 55.4% in the bean growing area, and the lowest at 64.0% and 24.3% in the faba bean growing area respectively. In this study, a total of 2714 fungal isolates were obtained from some legume plants and soil samples. The most common fungi isolated from root and soil samples were Fusarium spp., multinucleate Rhizoctonia (MNR), binucleate Rhizoctonia (BNR) and Pythium spp. respectively. Fusarium spp. were isolated at high rates from all the examined areas. MN Rhizoctonia and BN Rhizoctonia were isolated both from inner and coastal areas of the province, whereas Pythium spp. were isolated in costal areas, except for the Vezirköprü district which is situated in the inner area. When looking at the interactions among pathogens causing root rot, it was found the great majority of the samples (30.4%) isolated both Fusarium spp. and MNR-BNR group fungi, whereas Fusarium spp. and Pythium spp. were isolated together from 10.9% of the samples and MNR-BNR and Pythium spp. from only 1.5% of the samples.