A Threshold Value for Cutting Intensity in Relation to Fusarium Root Rot of Red Clover (Trifolium pratense L.)

Although cutting the foliage is known to increase Fusarium root rot severity in red clover (Trifolium pratense L.), no quantitative relationship has so far been determined. In this study, results from a number of greenhouse experiments, where plants were artificially inoculated with Fusarium avenaceum (Corda ex Fr.) Sacc., show a linear relationship between cutting intensity and Fusarium root rot in red clover, cv. ‘Hermes II’ an increased cutting intensity giving an increased root rot severity. Theoretically, a threshold value can be calculated beyond which no increase in root rot severity, compared to an uncut plant, should be expected.     

Effects of the Nicotiana debneyi black root rot resistance factor on agronomic and chemical traits in burley tobacco

Summary Lines isogenic or near isogenic for traits other than resistance to black root rot from Nicotiana debneyi were developed in eight cultivar backgrounds of burley tobacco (N. tabacum L.). For each cultivar background, a resistant and susceptible selection from the seventh backcross generation plus the recurrent parental cultivar were evaluated for ten agronomic and chemical traits. Resistant selections were statistically different from the susceptible entries for days to flower, total nitrogen content, and total alkaloid content. Also, resistant selections were consistently lower in yield, but the differences were statistically nonsignificant. Resistant selections were also taller in three families and in two families the resistant selections had wider leaves. Linkage of genetic material from N. debneyi with the resistance factor was suggested as the possible reason for differences between resistant and susceptible selections.     

The potential benefits from cultivar specific red clover -potato crop rotations

The aim of this study was to determine if endophytic bacteria could contribute to cultivar specific interactions between red clover (Trifolium pratense L.) and potatoes (Solanum tuberosum L.) in crop rotations. Endophytic bacteria were isolated from the roots of four red clover cultivars (AC Charlie, Altaswede, Marino and Tempus) grown in the field. Populations of bacteria from each cultivar were similar. The most abundant genus was Rhizobium, but species of Curtobacterium, Pseudomonas, and Xanthomonas were common to all cultivars. Plantlets of two potato cultivars, Russet Burbank and Shepody, were inoculated individually with the seven bacterial isolates most frequently recovered from each red clover cultivar, and grown in Magenta vessels for 6 wk. Significant differences were found for plant height, and wet weights of roots, shoots and their total. Potato cultivars differed for root wet weight only, while red clover cultivar, as a source of bacteria, had a significant effect on all traits except plant height. Differences among bacteria were significant for all traits except shoot wet weight. There was a significant interaction of potato cultivar by red clover cultivar. The potato cultivar Russet Burbank did best with bacteria from the red clover cultivar, Marino; and Shepody, with bacteria from Altaswede.     

Interaction between Sitona lepidus and red clover lines selected for formononetin content

Adult clover root weevil Sitona lepidus show a feeding preference for white clover Trifolium repens over red clover Trifolium pratense. The effects on S. lepidus of three red clover T. pratense lines, selected for high, medium, or low levels of the isoflavone formononetin in foliage, were compared in three experiments using white clover as a control. In a no‐choice slant board experiment, weevil larval weights were greater for larvae feeding on white clover roots than those feeding on roots of the red clovers. The effect of larval root herbivory on plant growth was similar for all four clovers. Following root herbivory, a large increase in root and shoot formononetin levels was observed in the high‐formononetin selection of red clover but little change in the low‐formononetin red clover. In a no‐choice experiment with sexually mature female adult weevils feeding on foliage of the four clovers, all the red clovers had increased weevil mortality. Female weevils eating the high‐formononetin red clover laid fewer eggs than weevils eating white clover. The red clover diet caused a large accumulation of abdominal fat and/or oil in the weevils, whereas weevils feeding on white clover did not accumulate fat/oil. When sexually immature adult weevils were given a choice of foliage from all four clovers, white clover was eaten preferentially, and the low‐formononetin red clover was preferred to the high‐formononetin red clover. The results suggest that formononetin and associated metabolites in red clover may act as chemical defences against adult S. lepidus and that distribution in forage legumes can be manipulated by plant breeding to improve root health.     

Inheritance of antagonistic properties and lytic enzyme activities in sexual crosses of Talaromyces flavus

Two wild-type strains and three benomyl-resistant mutants of the antagonistic ascomycete Talaromyces flavus were crossed in six combinations, two of which yielded hybrid cleistothecia. Parental strains and their ascospore progenies varied in several traits considered to play an important role in the capacity to control soilborne fungal pathogens: extracellular activities of glucose oxidase and cell-wall degrading enzymes, antibiosis towards Verticillium dahliae, and parasitism and biocontrol of Sclerotium rolfsii. A non-Mendelian quantitative mode of inheritance was found for β-1, 3-glucanase and chitinase activities but only the latter exhibited a normal frequency distribution. Some of the progenies exhibited higher glucanase and chitinase activities than those found in the parental strains. Progeny analysis for chitinase, glucanase, cellulase, and glucose oxidase activities revealed no genetic association between any two of these enzymes. Antibiosis was correlated with glucose-oxidase activity in one cross, but not in the other. The ability to reduce bean root rot caused by S. rolfsii was correlated with mycoparasitic activity against S. rolfsii sclerotia in one cross, but not in the other. One out of the 20 progenies tested was able to reduce bean root rot more effectively than its parent strains, thus demonstrating the feasibility of improving a biocontrol agent by conventional breeding.     

Biological control of clover rot on red clover by Coniothyrium minitans under natural and controlled climatic conditions

The ability of Coniothyrium minitans, contained in the commercial product Contans®WG, to control the development of clover rot in red clover, Trifolium pratense, was for the first time investigated in the field. Studies were performed on an established ley with a grass-clover mixture and on a newly sown pure red clover ley, both at a field site naturally infested with Sclerotinia trifoliorum. In the latter experiment the biocontrol agent was applied either prior to sowing or to growing seedlings. In addition, the ability of sclerotia of two S. trifoliorum isolates to cause disease in detached leaves was studied in a controlled environment. The effect of Contans®WG treatments at temperatures between +5 and +15°C and incubation periods of up to 7 weeks were included. Application of the biocontrol agent to the established ley during early summer, significantly reduced the number of groups of apothecia that developed during autumn in the following year in treated plots, compared to untreated plots. Twice as many red clover plants of the cultivar SW Torun survived in the pure red clover stand experiment the year after Contans®WG application as in the untreated plots, irrespective of how the agent was applied. In the laboratory studies, administering biocontrol treatments to sclerotia significantly reduced disease scores in the detached leaves at all temperatures at an exposure time of 7 weeks. Shorter incubation periods did not always negatively affect sclerotial viability.     

The inheritance of preference for strains of Rhizobium trifolii by white clover (Trifolium repents)

The preference or selection of Rhizobium strains by both parental and F₁ white clover plants was studied using antibiotic resistant mutants of Rhizobium trifolii. Highly significant differences were found between crosses in their preference for 75 str. Progenies from crosses between plants in which the preference for 75 str was 90–100% had 85% of the nodules inhabited by that strain. Progenies of crosses between plants in which the preference for 75 str was less than 50% had only 13% of their nodules inhabited by 75 str. Intermediate values were found for progenies from other crosses. The inheritance for this particular character was clearly additive and without any dominance or maternal effect. The results are discussed from the point of view of improving symbiotic nitrogen fixation by breeding white clover for uniform preference for effective strains of R. trifolii.     

Response of subterranean clover cultivars to root rot fungi

Twelve widely grown cultivars of subterranean clover (Trifolium subterraneum) were screened both under controlled environment conditions for their resistance to five fungi commonly associated with root rot and under field conditions for their resistance to natural root infections. All cultivars showed decreased seedling survival (particularly from Pythium irregulare and Rhizoctonia solani), tap and lateral root rot (particularly from Fusarium avenaceum, P. irregulare, and R. solani) and reduced plant size (particularly from R. solani and P. irregulare). Individual cultivars generally differed in their response to the five pathogens and for any one pathogen there was generally a range of cultivar susceptibilities. Cultivars with the best resistance to individual root pathogens were identified. The results for the five individual pathogens under controlled conditions only showed correlation with field data for some of the parameters compared.     

In vitro selection at cellular level for red rot resistance in sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> sp.)

In the present study, in vitro selection technique using pathogen culture filtrate of Colletotrichum falcatum Went was employed with the aim to identify associations (if any), between selection at the cellular and plant level for red rot resistance in sugarcane (Saccharum sp.). Five to eight months old sugarcane calli of genotypes CoJ 88 and CoJ 64 were screened in vitro against pathogen culture filtrate for two selection cycles. Effect of pathogen culture filtrate on callus survival and/or proliferation was observed to be directly related to its concentration in the selection media. Calli survived and exhibited further proliferation at 5, 10 and 15% v/v pathogen culture filtrate concentrations whereas, at higher concentrations (20 and 25% v/v) proliferation was completely inhibited. Shoot regeneration percent was higher in calli selected on 5% pathogen culture filtrate concentration than those selected on 10 and 15% concentrations. In vivo screening of field transferred somaclones against two pathtypes (Cf 03 and Cf 08) showed considerable variation for red rot resistance. Somaclones regenerated from resistant and/or tolerant calli exhibited better resistance than the parental genotypes. The results indicated that in vitro selection for red rot resistance was effective and expressed when somaclones were screened in the field. This indicated a positive association between in vitro and in vivo methods of selection for disease resistance in sugarcane.     

In vitro selection for 2,4-D tolerance in red clover

Summary In vitro, selection is a viable method of selecting herbicide-tolerant crops. This research was to evaluate in vitro selection techniques for enhancing 2,4-D [(2,4-dichlorophenoxy) acetic acid] tolerance in red clover (Trifolium pratense L.). In vivo and in vitro responses to 2,4-D of eight diverse red clover populations were correlated (r=0.77), justifying in vitro selection for 2,4-D tolerance. Suspension cultures of a red clover genotype capable of regeneration were plated onto agar-based nutrient media supplemented with 0.18 mM 2,4-D for selection experiments. After two cycles of selection, 16 2,4-D tolerant callus lines were identified based on visual growth assessment. These lines were evaluated for 2,4-D tolerance (based on 2,4-D content), using a 2,4-D bioassay procedure which consisted of placing selected callus tissue pieces on top of oat (Avena sativa L.) coleoptile or internode sections. The relative amount of 2,4-D in the callus tissue was estimated by the amount of oat section elongation after 24 h. Two of the more tolerant callus lines had 61% and 83% less 2,4-D in their tissues than the susceptible control tissue. These studies indicated that in vitro selection can enhance the levels of 2,4-D tolerance in red clover callus tissue.Florida Agricultural Experiment Station Journal Series No. 8943     

The Role of Polyphenol Oxidase and Peroxidase in Potato Tuber Resistance to Soft Rot Caused by Erwinia carotovora

Tubers from somatic hybrids produced by protoplast fusion between Solanum brevidens, a diploid, non-tuber-bearing wild species, and a tetraploid S. tuberosum showed resistance to decay caused by soft rot Erwinia. Tubers of the S. tuberosum fusion parent and potato cultivar Russet Burbank are susceptible to bacterial soft rot. Tubers of somatic hybrids indicated higher levels of activities of polyphenol oxidase and peroxidase than tubers of the parental line of S. tuberosum and cultivar Russet Burbank. This is true for intact tubers and also for injured or inoculated tubers. Polish commercial potato cultivars indicated a higher susceptibility to soft rot than somatic hybrids. However, there were some differences in susceptibility to soft rot between Polish commercial potato cultivars, only slight differences were observed in the activities of the polyphenol oxidase and peroxidase between Polish cultivars. A relation between soft rot resistance and the activity of each enzyme was not found for intact, injured or inoculated tissue of commercial cultivars. On the contrary, the activities of both enzymes were significantly higher in the periderm than in the medullary tissue of somatic hybrids, the parental line and the commercial cultivars.     

Characterization of novel microsatellite loci for red clover (Trifolium pratense L.) from enriched genomic libraries

Twenty‐seven polymorphic microsatellite markers were isolated from red clover (Trifolium pratense). Allelic variability and cross‐species amplification were assessed on 24 red clover and eight white clover (Trifolium repens) genotypes. The number of alleles detected in red clover ranged from two to 25. Observed and expected heterozygosities were high with average values of 0.71 and 0.88, respectively. Five of the 27 loci were also successfully amplified from white clover, where two to 13 alleles were detected. These highly polymorphic microsatellite loci provide powerful tools for population genetic studies as well as for marker‐assisted selection in this important forage legume species.     

Isoflavone Content in Subterranean Clover Germplasm from Sardinia

Subterranean clover (Trifolium subterraneum) is an important pasture legume, and Sardinia is known as a major centre of diversification of this species. As other legumes, this clover produces biologically active flavonoids including the subclass of isoflavones that are natural phytoestrogens with positive health effects. Present sources of isoflavones for medical/nutraceutical treatments are red clover (Trifolium pratense) and soybean (Glycine max). This study assessed the content and composition of flavonoids in 14 subterranean clover genotypes from Sardinia, grown ex‐situ in comparison with two red clover ecotypes, to acquire information on the potential of the species as an alternative source of isoflavones for possible exploitation. Twenty compounds were tentatively identified across the two clovers after HPLC and LC/ESI‐MS analyses, including clovamide, four flavonols, and 15 isoflavones. Most compounds were present as glucosides or glucosyl malonates. Subterranean clover extracts mainly comprised of derivatives of the isoflavones genistein, biochanin A, and formononetin. Compared to red clover, subterranean clover had higher content of total isoflavones and lower concentration of total flavonols. The isoflavone concentration in subterranean clover was higher than literature data for soybean or red clover. The existing genotypic variation warrants the possibility of selecting varieties with high isoflavone concentration for nutraceutical or pharmaceutical purposes.     

Root Rot of Trifolium subterraneum Induced by Fusarium solani

A fungus disease of Trifolium subterraneum, previously unknown in South Africa, was identified as Fusarium root rot caused by F. solani (Mart.) Sacc. Diseased plants were severely stunted. The characteristic internal root discolouration observed in the field, is described. The disease occurred mainly between Swellendam and Humansdorp in the southern and south-eastern Cape Province. The subterranean clover fields inspected were between 95 and 100 % infected with the root rot disease. T. repens and T. pratense were also susceptible.     

Fungicidal control of damping-off and seedling root rot in Brassica species caused by Rhizoctonia solani in the growth chamber*

Eight fungicides (benodanil, carboxin, cyproconazole, fenpropimorph, fur-mecyclox, iprodione, pencycuron and tolclofos-methyl) were evaluated, under growth chamber conditions, as seed treatments against pre-emergence damping-off and post-emergence seedling root rot in six Brassica species. Five cultivars of B. rapa, four cultivars of B. juncea, four cultivars of B. napus and one cultivar/ strain from each of B. carinata, B. nigra and B. oleracea were grown in soilless mix infested with an isolate of Rhizoctonia solani AG-2-1. B. nigra and B. juncea were considerably less susceptible to R. solani than the four other species. Cyproconazole at 0.05-0.1 g a.i./kg seed and the other fungicides at 2–4 g a.i./ kg seed provided almost complete control of pre-emergence damping-off in most Brassica species and their cultivars. Their efficacy varied against the post-emergence seedling root rot. Furmecyclox, iprodione, tolclofos-methyl and pencycuron consistently gave good control of seedling root rot in all Brassica species and their cultivars. Benodanil and fenpropimorph provided moderate control, and carboxin and cyproconazole gave poor control against root rot. Efficacy of carboxin, cyproconazole, benodanil and fenopropimorph against seedling root rot varied significantly (P ≤ 0.05) among cultivars within a Brassica species.     

Distinguishing between metabolically active and inactive roots by combined staining with 2,3,5-triphenyltetrazolium chloride and image colour analysis

The objective of the present work was to develop a method to distinguish between metabolically inactive and active parts of plant roots. White clover (Trifolium repens L.) roots were stained with 2,3,5-triphenyltetrazolium chloride (TTC) followed by root colour classification with an interactive scanner-based image analysis programme (WinRHIZO). Roots inactivated by boiling were unstained and pale brown, whereas fresh samples with predominantly metabolically active roots turned dark red, red or pale red after staining. A small amount of very young, presumable active roots (0.8% of total active root length) failed to stain red with TTC. The colour analysis of inactive and active roots was based on four colour classes for boiled roots and seven classes for fresh roots, respectively, as defined upon visual examination of images. Pixel colours falling outside the defined classes were allocated to the nearest defined class – an option that increased objectivity and stability and reduced the required number of colour classes. For the fresh white clover roots, 75–86% of the total root length was determined as active, while 3–7% of the boiled roots fell into the same category. The percentage of total root length measured by WinRHIZO that was identified as metabolically active was linearly correlated with the percentage of fresh roots in mixtures of fresh and boiled roots (R²=0.99). Colour classes chosen à priori from one experiment could be used to distinguish fairly satisfactorily between active and inactive roots of another white clover cultivar grown under other conditions, but failed to classify activity in ryegrass (Lolium multiflorum Lam.) root samples. In the latter case, colour classes needed to be re-defined in order to produce reliable data. Our work shows that WinRHIZOs colour identification sub-module provides a new promising tool to classify root activity as identified after staining with TTC, but colour classes must be carefully evaluated on every new occasion.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Root Dynamics of Cultivar and Non‐Cultivar Population Sources of Two Dominant Grasses during Initial Establishment of Tallgrass Prairie

Dominance of warm‐season grasses modulates tallgrass prairie ecosystem structure and function. Reintroduction of these grasses is a widespread practice to conserve soil and restore prairie ecosystems degraded from human land use changes. Seed sources for reintroduction of dominant prairie grass species include local (non‐cultivar) and selected (cultivar) populations. The primary objective of this study was to quantify whether intraspecific variation in developing root systems exists between population sources (non‐cultivar and cultivar) of two dominant grasses (Sorghastrum nutans and Schizachyrium scoparium) widely used in restoration. Non‐cultivar and cultivar grass seedlings of both species were isolated in an experimental prairie restoration at the Konza Prairie Biological Station. We measured above‐ and belowground net primary production (ANPP and BNPP, respectively), root architecture, and root tissue quality, as well as soil moisture and plant available inorganic nitrogen (N) in soil associated with each species and source at the end of the first growing season. Cultivars had greater root length, surface area, and volume than non‐cultivars. Available inorganic N and soil moisture were present in lower amounts in soil proximal to roots of cultivars than non‐cultivars. Additionally, soil NO₃–N was negatively correlated with root volume in S. nutans cultivars. While cultivars had greater BNPP than non‐cultivars, this was not reflected aboveground root structure, as ANPP was similar between cultivars and non‐cultivars. Intraspecific variation in belowground root structure and function exists between cultivar and non‐cultivar sources of the dominant prairie grasses during initial reestablishment of tallgrass prairie. Population source selection should be considered in setting restoration goals and objectives.     

Comparison of Screening Methods for Resistance to Fusarium Root Rot in Common Beans (Phaseolus vulgaris L.)

Root rot, caused by Fusarium solani f. sp. phaseoli, is one of the main root diseases impacting production of common beans throughout the world. Because resistance of common beans to root rot is a quantitative trait that is strongly influenced by environmental factors, reproducible methods to screen bean plants for resistance to root rot are critical to the selection process. In this study, we adapted the inoculum layer method (ILM) developed for screening soybeans for resistance to Phytophthora rot and compared it with the traditional liquid inoculum method (LIM) for screening common beans for resistance to Fusarium root rot. In addition, two methods of evaluating resistance using the ILM were compared. The most significant Pearson correlation coefficient between trials involving 80 recombinant inbred lines was achieved with the ILM and counting discoloured vascular bundles in the lower stem (r_p = 0.7113***) compared to rating the discoloration on root and hypocotyl (r_p = 0.5555***). The traditional (LIM) screening method and rating the discolouration on roots resulted in a non‐significant correlation between trials (r_p = 0.1084).     

Inheritance of Resistance to Pratylenchus penetrans in Alfalfa

Fifty-two alfalfa (Medicago sativa L.) clones, randomly selected from the cultivar Baker and the experimental line MNGRN-4, were evaluated for resistance (based on nematode reproduction) to Pratylenchus penetrans in growth chamber tests (25 C). Twenty-five clones, representing the range of nematodes and eggs per plant, were selected and retested. Four moderately resistant and two susceptible alfalfa clones were identified. Inheritance of resistance to P. penetrans was studied in these six clones using a diallel mating design. The S₁, Fl, and reciprocal progenies differed for numbers of nematodes and eggs per g dry root and for shoot and root weights (P < 0.05). Resistance, measured as numbers of nematodes in roots, was correlated between parental clones and their S₁ families (r = 0.94), parental clones and their half-sib families (r = 0.81), and S₁ and half-sib families (r = 0.88). General combining ability (GCA) effects were significant for nematode resistance traits. Both GCA and specific combining ability (SCA) effects were significant for plant size traits, but SCA was more important than GCA in predicting progeny plant size. Reciprocal effects were significant for both nematode resistance and plant size traits, which may slow selection progress in long-term selection programs. However, the GCA effects are large enough that breeding procedures that capitalize on additive effects should be effective in developing alfalfa cultivars with resistance to P. penetrans.