A Rapid and Efficient Method for In Vitro Screening of Taro for Leaf Blight Disease Caused by Phytophthora colocasiae

Taro leaf blight caused by Phytophthora colocasiae presents the single biggest constraint for taro cultivation globally. To accelerate breeding and selection for disease resistance to leaf blight, it is important to develop bioassays which could differentiate resistant and susceptible cultivars efficiently. In this study, thirty taro accessions and four released cultivars were evaluated for resistance to leaf blight using a modified floating leaf disc assay. A novel method for mass production of P. colocasiae zoospores was developed and used as inoculum for the assay. There were significant (P < 0.05) differences among accessions in their response to P. colocasiae infection in the detached leaf assay. The accessions could be efficiently classified into various resistance groups based on a 0–4 score. Also, the assay results were consistent with the field evaluation scores of taro accessions. Thus, this study reports the development of a rapid, simple and repeatable assay that can be used to screen large numbers of taro cultivars for resistance to P. colocasiae.     

Evaluation of fungicides on Indian isolates of Phytophthora colocasiae causing leaf blight of taro

Leaf blight caused by Phytophthora colocasiae is the most destructive disease affecting taro (Colocasia esculenta) worldwide including India. Fungicides (primarily metalaxyl) remain as an important strategy to manage taro leaf blight in India over decades. It is important to monitor isolate sensitivity to identify build-up of fungicide resistance and thereby modify fungicide usage strategies. P. colocasiae isolates representing four different geographical regions of India were evaluated for their sensitivity to metalaxyl and three other commercially available fungicides viz. Samarth, Biofight and Akoton by poisoned media technique. All the isolates tested were sensitive to metalaxyl, nevertheless there is an increase in the effective concentration compared to the previous reports. Among the other fungicides, Samarth was found to be superior in completely inhibiting mycelial growth at 0.05% followed by Biofight at 1%. Metalaxyl and Akoton^® shared a common inhibitory concentration at 2%. The most effective fungicide determined by the in vitro method was evaluated in vivo for studying the pattern of inhibition before and after the disease development in detached taro leaf. The results of the study revealed that build-up on metalaxyl resistance in P. colocasiae is in its course and caution should be taken while administering against taro leaf blight. Fungicide Samarth could be used as an alternative to metalaxyl for management of taro leaf blight.     

Antioxidative enzymes and isozymes analysis of taro genotypes and their implications in <Emphasis Type="Italic">Phytophthora</Emphasis> blight disease resistance

Assessment of the differential expression of antioxidative enzymes and their isozymes, was done in 30 day-old ex vitro raised plants of three highly resistant (DP-25, Jhankri and Duradim) and one highly susceptible (N-118) genotypes of taro [Colocasia esculenta (L.) Schott]. Antioxidative enzymes were assayed in the ex vitro plants, 7 days after inoculation with the spores (15,000 spores ml⁻¹ water) of Phytophthora colocasiae Raciborski to induce taro leaf blight disease. Uninoculated ex vitro plants in each genotype were used as control. The activity of superoxide dismutase (SOD) and guaiacol peroxidase (GPX) increased under induced blight condition when compared with control. Increase in antioxidative enzymes was more (67–92%) in the resistant genotypes than that (21–29%) of the susceptible genotype. The zymograms of SOD and GPX in the resistant genotypes, with pathogenic infection, showed increased activity for anodal isoform of SOD and increased expression and/or induction of either POX 1 or POX 2 isoforms of GPX. In susceptible genotype, expression of the above isoforms was faint for SOD and nearly absent for GPX under both blight free and induced blight conditions. Induction and/or increased activity of particular isoform of SOD and GPX against infection of Phytophthora colocasiae in the resistant genotypes studied led to the apparent conclusion of linkage of isozyme expression with blight resistance in taro. This might be an important criterion in breeding of taro for Phytophthora leaf blight resistance.     

Variations in morphological and molecular characteristics of Phytophthora colocasiae population causing leaf blight of taro in Ghana

Abstract

The development of sustainable and appropriate management strategy to control leaf blight of taro depends on a good understanding of the population structure of its causal pathogen. Morphological and molecular techniques were used to characterise the population of Phytophthora colocasiae obtained from different locations in Ghana. Based on colony growth habit, the isolates were grouped into four morphogroups and expressed significant differences in colony diameter. Molecular analysis revealed polymorphism among the isolates. A dendrogram produced using unweight pair group method with arithmetic mean generated grouped isolates of P. colocasiae into six clusters. Results obtained proved the existence of variation in the causal pathogen of taro leaf blight disease in Ghana. This provides an important basis for developing suitable management strategies against leaf blight of taro caused by P. colocasiae based on good integrated approach.

     

Storage rots of taro (Colocasia esculenta) in the British Solomon Islands

Several fungicides chosen for their ability to control the pathogens previously isolated from stored corms of taro failed to prevent severe rotting. This result led to a reappraisal of the organisms involved in the initial stages of decay. Isolations made from stored corms during the first 5 days showed that Phytophthora colocasiae and Pythium splendens were the dominant fungi in the rots. Later Botryodiplodia theobromae rapidly colonized the corms to complete the decay. Attempts to reduce losses by leaving petiole bases, cormels and roots attached only succeeded in delaying infection by a few days. Corms placed in soil in well-drained pits stored relatively well up to 4 wk without impaired taste. Fungal rots were completely eliminated in corms stored in the soil but bacterial rots caused by Erwinia chrysanthemi were responsible for some decay.     

Changes in Phenolics, Polyphenol Oxidase and its Isoenzyme Patterns in Relation to Resistance in Taro against Phytophthora colocasiae

The effect of Phytophthora leaf blight disease, caused by Phytophthora colocasiae Raciborski, on the accumulation of phenolics and polyphenol oxidase (PPO) activity in ex vitro plants was studied in three resistant (DP‐25, Duradim and Jhankri) and one susceptible (N‐118) genotypes of taro [Colocasia esculenta (L). Schott]. The inoculation of taro leaves with P. colocasiae spores resulted in a quantitative change in both biochemical parameters and induction of PPO isoforms in resistant genotypes. The amount of phenolics was increased owing to blight by 68.02%, 58.87%, 52.67% and 11.50% in DP‐25, Duradim, Jhankri and N‐118, respectively. The per cent increase in PPO under stress over non‐stress condition was also highest in DP‐25 (49.14%) followed by Duradim (41.56%), Jhankri (40.55%) and N‐118 (17.08%). The resistant genotypes showed higher activity of PPO as compared with susceptible ones, which was reflected through its banding pattern in isoenzyme analysis, detecting four different isoforms. The intensity of the bands was higher in the resistant genotypes than in susceptible N‐118. The appearance of high intensity bands and/or reduction in the intensity of particular isoform(s) in the zymograms of all the three resistant taro genotypes studied, led to the apparent conclusion of linking PPO isoenzyme expression with blight resistance in taro. The blight incidence (per cent leaf infection and leaf area infection) was lower in the resistant genotypes than in susceptible, N‐118. The yield reduction owing to blight was below 20% in DP‐25, Jhankri and Duradim, while the same was more than 40% in N‐118. The phenolics and PPO activity was negatively correlated with disease incidence and yield reduction owing to blight. Based on the results of disease incidence, biochemical contents and yield, the pattern of stress tolerance was DP‐25 > Duradim > Jhankri > N‐118. The studied parameters, i.e. phenolics and PPO could be used as biochemical markers for leaf blight stress tolerance studies in taro.     

Effect of VAM inoculum carry-over on the successive cropping of maize and mungbean

A field study to determine the endomycorrhizal inoculum carry-over effect of the first crop [maize inoculated with Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe] on the succeeding crop (mungbean) was carried out in fumigated and nonfumigated acidic soil (pH 5.3) with moderate extractable P (Olsen 23 ppm). G. mosseae inoculation increased maize dry matter and grain yield over the uninoculated control in the nonfumigated soil. The maize inoculation failed to carry the effective inoculum over to the mungbean crop planted immediately after maize harvest and thus did not increase root colonization and grain yield of the succeeding crop. Fresh inoculation of the mungbean with G. mosseae increased grain yield over the uninoculated control.     

Effects of preplant inoculation of apple (Malus domestica Borkh.) with arbuscular mycorrhizal fungi on population growth of the root-lesion nematode,Pratylenchus penetrans

Six species of arbuscular mycorrhizal (AM) fungi (Glomus aggregatum, G. clarum, G. etunicatum, G. intraradices, G. mosseae and G. versiforme) were evaluated, in three greenhouse experiments, for their effects on reproduction of the root-lesion nematode, Pratylenchus penetrans, and growth of Ottawa 3 apple rootstock. Glomus mosseae increased total dry weights of nematode-inoculated and non-inoculated rootstock in all three greenhouse experiments, and G. intraradices increased dry weights in two of three greenhouse experiments. Plants inoculated with G. mosseae generally supported fewer P. penetrans per gram of root than plants inoculated with other AM fungi, but did not differ significantly from the controls in any greenhouse experiment. Colonization of roots by AM fungi was reduced by P. penetrans at initial inoculum densities greater than 250 nematodes/L soil. In field trials, preplant inoculation with either G. intraradices or G. mosseae increased rootstock growth and leaf concentrations of P, Mg, Zn and Cu in fumigated plots but not in non-fumigated plots, indicating that colonization by native AM fungi in non-fumigated plots may have been sufficient for adequate nutrient acquisition. The abundance of vesicles and arbuscules was greater in roots of plants inoculated with AM fungi before planting than in roots of non-inoculated plants, in both fumigated and non-fumigated plots. P. penetrans per gram of root and per 50 ml soil were significantly lower for G. mosseae- inoculated plants than for non-inoculated plants in fumigated soil but not in non-fumigated soil.     

Isozyme and PCR-based genotyping of epidemic Phytophthora colocasiae associated with taro leaf blight

The Oomycetous fungus Phytophthora colocasiae causing leaf blight of taro is widely distributed in India. Wide geographic range or sexual recombination provides genetic differentiation within this species. To determine how genetic variation is partitioned in P. colocasiae, 14 isolates were isolated from different regions of India, where the incidence of leaf blight is great. Molecular and biochemical techniques were employed for assessing and exploiting the genetic variability among isolates of P. colocasiae. Seven polymorphic enzyme systems revealed 23 isozyme patterns, each uniquely characterised by the presence or absence of electromorphs. Further, 10 oligodeoxynucleotide primers were selected for random amplified polymorphic DNA (RAPD) assays, which resulted in 123 polymorphic bands for 10 isolates of P. colocasiae. The data were entered into a binary matrix and a similarity matrix was constructed using a DICE similarity (SD) index. A UPGMA cluster based on SD values was generated using a NTSYS computer program. Shannon's index was used to partition genetic diversity. Similarly, isozymes and RAPDs yielded high estimates of genetic variability. Genetic diversity estimates via isozyme and RAPD pattern indicated 78.26% and 100%, respectively, total diversity among populations. This type of genetic variation in P. colocasiae indicates that variation due to asexual and/or possibly infrequent sexual mechanisms is possible and that genetic differentiation has taken place as a result of geographic isolation. The presence of larger than expected RAPD variation in isolates of P. colocasiae and the presence of distinct different zymotypes among these isolates suggests that genetic recombination (or less likely hybridisation) is at least possible in this fungus and that geographic differentiation has taken place. Even isolates obtained from the same habitat have different RAPD patterns, indicating that many populations of this fungus are made up of more than one genet and that few are derived clonally.     

Resistance in apple to shoot infection by Venturia inaequalis

Eight apple genotypes, including cultivars and breeding selections resistant and susceptible to Venturia inaequalis on foliage, were screened for shoot infection and the development of wood pustules following inoculation of shoot tips of 1–year maiden trees in the greenhouse. Where genotypes were highly resistant in terms of foliar symptoms (cvs Prima and Gavin), no shoot infection was observed. Where genotypes were highly susceptible in terms of foliar symptoms, then shoot tissue was either resistant (cv. Shinko) or susceptible (cv. Starking). In one experiment, shoot tips were inoculated sequentially as shoots extended. No pustules developed where inoculations were made later than May. In a further experiment, two leaf internode positions were inoculated on one occasion. Pustules were only observed where inoculations were made above the youngest unrolled leaf. The results suggested that shoot tips were more susceptible during early extension growth of the shoot. Pustules were noted in abundance on petioles of susceptible cultivars, and these probably contributed to early leaf abscission.     

Effects of spur blight (Didymella applanata) and premature defoliation on axillary buds and lateral shoots of red raspberry

Nodal lesions typical of natural infections of Didymella applanata were produced on young raspberry canes when wounded petioles were inoculated with mycelium. The buds which developed in the axils of these inoculated petioles were smaller and less likely to produce lateral shoots than those in the axils of delaminated or undamaged petioles, but only when the petioles were inoculated in the field before the end of July. This effect was more pronounced in cv. Mailing Jewel than in cv. Glen Clova which suggests that the latter is more tolerant of D. applanata. Sprays to control D. applanata in the cropping region of canes will probably be effective in Scotland only if applied before the end of July.     

Salt tolerance of rice cultivars

Summary The dry matter production and the concentration of nutrients in rice (Oryza sativa L.) cultivars from soil adjusted to different levels of salinity were evaluated under a greenhouse conditions. Soil salinity levels were produced by applying 0.34 mol l^–1 solution of NaCl which resulted in the following levels, control (0.29), 5, 10 and 15 dS m^–1 conductivity of saturation extract. The effect of salinity on dry matter production varied from cultivar to cultivar.The concentrations of P and K in the tops of rice cultivars decreased with increasing soil salinity. But the concentrations of Na, Zn, Cu and Mn increased.Significant varietal differences were found in relation to salinity tolerance. Based on dry matter yield reduction, rice cultivars were classified as tolerant, moderately tolerant, moderately susceptible or susceptible.     

The genetic structure of taro: a comparison of RAPD and isozyme markers

Germplasm characterization and evolutionary process in viable populations are important links between the conservation and utilization of plant genetic resources. Here, an investigation is made, based on molecular and biochemical techniques for assessing and exploiting the genetic variability in germplasm characterization of taro, which would be useful in plant breeding and ex situ conservation of taro plant genetic resources. Geographical differentiation and phylogenetic relationships of Indian taro, Colocasia esculenta (L.) Schott, were analyzed by random amplified polymorphic DNA (RAPD) and isozyme of seven enzyme systems with specific reference to the Muktakeshi accession, which has been to be proved resistant to taro leaf blight caused by P. colocasiae. The significant differentiations in Indian taro cultivars were clearly demonstrated by RAPD and isozyme analysis. RAPD markers showed higher values for genetic differentiation among taro cultivars and lower coefficient of variation than those obtained from isozymes. Genetic differentiation was evident in the taro accessions collected from different regions of India. It appears that when taro cultivation was introduced to a new area, only a small fraction of genetic variability in heterogeneous taro populations was transferred, possibly causing random differentiation among locally adapted taro populations. The selected primers will be useful for future genetic analysis and provide taro breeders with a genetic basis for selection of parents for crop improvement. Polymorphic markers identified in the DNA fingerprinting study will be useful for screening a segregating population, which is being generated in our laboratory aimed at developing a taro genetic linkage map.     

Effects on plant growth of inoculation of stored stripmining topsoil in North Dakota with mycorrhizal fungi contained in native soils

Stored topsoil from stripmining operations in western North Dakota was inoculated with mycorrhizal fungi contained in native prairie soil. The effects on plant mycorrhizal infection percentage, growth as shoot dry weight, and phosphorus uptake were determined. The studied topsoil piles were found to contain little or no vesicular arbuscular mycorrhizal (VAM) fungal inoculum at a depth of 120 cm. The inoculum soil was mixed into the stored soil at rates of 10% and 1%, or surface-applied at 1%. In control pots, sterilized inoculum soil was used. Corn plant (Zea mays) bioassays were used. After 30 days growth the percent VAM fungal infection of the test plants increased with both the 10% and 1% soil inocula. Phosphorus concentrations were generally increased by inoculation with 10% soil mixtures but not 1%. Shoot dry weights of the plants were not measurably different between 10% and 1% inoculation. However, when the plant growth period was increased to 60 days, all three parameters were increased over the check plants. When the inoculum was not mixed into the soil, but layered on the surface, there were no differences in any of the parameters.     

Host specificity of Puccinia spegazzinii (Pucciniales: pucciniaceae), a biological control agent for Mikania micrantha (Asteraceae) in Australia

The biology and host specificity of the rust fungus Puccinia spegazzinii, a biological control agent for Mikania micrantha, was studied in a quarantine laboratory in Brisbane, Australia. Nineteen plant species, all from the family Asteraceae were inoculated during host specificity trials. Spore formation occurred only on M. micrantha and no other plant was infected. The first signs of infection (white spots on leaves, petioles and stems) occurred 7 days after exposure to inoculum, becoming yellow by day 11. Mature pustules containing teliospores had developed by 20 days. Results obtained here support testing in other countries showing that P. spegazzinii is host specific to M. micrantha and no that other plant in Australia would be at risk if it were to be released in the environment. Recommendations on assessing pathogens to improve trials were proposed. Counting infected plants, plant parts and pustules formed can demonstrate that all test plants would have been adequately exposed to inoculum and thus increase the confidence in host specificity of pathogens in weed biological control.     

Chemical alteration of the rhizosphere of the mycorrhizal-colonized wheat root

Plexiglass pot growth chamber experiments were conducted to evaluate the chemical alterations in the rhizosphere of mycorrhizal wheat roots after inoculation with Glomus intraradices [arbuscular mycorrhizal fungus (AMF)]. Exchange resins were used as sinks for nutrients to determine whether the inoculated plant can increase the solubility and the uptake of P and micronutrients. Treatments included: (1) soil (bulk soil); (2) AMF inoculation no P addition (I–P); (3) no inoculation with no P addition (NI–P); (4) AMF inoculation with addition of 50 mg P (kg soil)^–1 (I+P), and (5) no inoculation with addition of 50 mg P (kg soil)^–1 (NI+P). The AMF inoculum was added at a rate of four spores of G. intraradices (g soil)^–1. The exchange resin membranes were inserted vertically 5 cm apart in the middle of Plexiglass pots. Spring wheat (Triticum aestivum cv. Len) was planted in each Plexiglass pot and grown for 2 weeks in a growth chamber where water was maintained at field capacity. Rhizosphere pH and redox potential (Eh), nutrient bioavailability indices and mycorrhizal colonization were determined. Mycorrhizal inoculation increased the colonization more when P was not added, but did not increase the shoot dry weight at either P level. The rhizosphere pH was lower in the inoculated plants compared to the noninoculated plants in the absence of added P, while the Eh did not change. The decrease in pH in the rhizosphere of inoculated plants could be responsible for the increased P and Zn uptake observed with inoculation. In contrast, Mn uptake was decreased by inoculation. The resin-adsorbed P was increased by inoculation, which, along with the bioavailability index data, may indicate that mycorrhizal roots were able to increase the solubility of soil P.     

Increasing phosphorus concentration in seed of annual pasture legume species increases herbage and seed yields

In glasshouse experiments with low levels of soil applied phosphorus (P), yields of four annual pasture legumes (Medicago polymorpha, Trifolium subterraneum, T. balansae, Ornithopus compressus) increased with increasing P concentration in the seed. In a further experiment, M. polymorpha cv. Serena was grown at the same plant density from seed of two P concentrations and two seed sizes when two levels of finely ground superphosphate were applied to the soil. Higher P concentrations in the seed increased yields of dried tops by about 30% for the first harvest (21 days), 20% for the second harvest (52 days), and 9% at maturity (103 days), and seed yields by 11%. Larger seeds increased yields of dried tops by between 6–46% for the first two harvests but at maturity yields of dried tops and seed were unaffected by seed size. None of the interactions were statistically significant (P>0.05), except for the first harvest when two interactions (P concentration in the seed × seed size (i.e. P content in seed), and P applied to the soil × P concentration in the seed × seed size) were significant at P<0.05 level. In a field experiment, Trifolium subterraneum clover seed (two cultivars) of the same size but with two different P concentrations was sown at the same plant density and two levels of granulated (0.2–5 mm) superphosphate were applied to the soil surface. The higher level of superphosphate increased dried herbage yields of the dense clover swards by three- to four-fold 90 and 120 days after sowing. The higher P concentration in the seed increased yields of dried herbage by between 50 to 25%, depending on the level of P applied to the soil and the harvest date.     

Effects of organomercury treatment of wheat seed on Fusarium seedling disease in inoculated soil

In growth room experiments, treatment of wheat seed with phenyl mercuric acetate significantly reduced seedling disease caused by soil-borne Fusarium culmorum, F. nivale and F. avenaceum in inoculated soil. Seed treatment improved the low germination caused by F. culmorum, but did not affect the reduced seedling vigour caused by F. nivale. Seed treatment was normally as effective in conditions favouring disease as it was in less favourable conditions. Conditions favouring disease in soil inoculated with F. culmorum were high inoculum level, deep sowing and dry soil.     

Soil fumigation and phosphorus supply affect the formation of Pisolithus-Eucalyptus urophylla ectomycorrhizas in two acid Philippine soils

To examine the effects of microbial populations and external phosphorus supply of two Philippine soils on mycorrhizal formation, Eucalyptus urophylla seedlings were inoculated with two Pisolithus isolates and grown in fumigated, reinfested and unfumigated soil fertilized with four rates of phosphorus. The Pisolithus isolates used were collected from under eucalypts in Australia and in the Philippines. Soils were infertile acid silty loams collected from field sites in Pangasinan, Luzon and Surigao, Mindanao.Significant interaction was observed between inoculation, soil fumigation and phosphorus supply on mycorrhizal formation by the Australian isolate in Surigao soil but not in Pangasinan soil. Soil fumigation enhanced mycorrhizal formation by the Australian isolate but did not affect root colonization by the Philippine isolate. Root colonization by the Australian isolate was highest in the reinfested soil while for the Philippine isolate it was highest in the unfumigated soil. The Australian isolate was more effective than the Philippine isolate in promoting growth and P uptake of E. urophylla seedlings in both soils. Total dry weight and P uptake of E. urophylla seedlings inoculated with the Australian isolate were maximum in fumigated and in the reinfested Pangasinan and Surigao soils supplied with 8 mg P kg^-1 soil. In the unfumigated soil, growth of seedlings inoculated with the Australian isolate was significantly reduced. Seedlings inoculated with the Philippine isolate had the largest dry weights and P contents in unfumigated Pangasinan and Surigao soils supplied with 8 mg P kg^-1 soil.These results indicate that the performance of the Australian Pisolithus isolate was markedly affected by biological factors in unfumigated soil. Thus, its potential use in the Philippines needs to be thoroughly tested in a variety of unfumigated soils before its widespread use in any inoculation programme.