Diversity study on Sclerotinia trifoliorum Erikks., the causal agent of clover rot in red clover crops (Trifolium pratense L.)

Since the 16th century, red clover has been an important crop in Europe. Since the 1940s, the European areal of red clover has been severely reduced, due to the availability of chemical fertilizers and the growing interest in maize. Nowadays there is a growing interest in red clover again, although some setbacks still remain. An important setback is the low persistence of red clover crops. Clover rot, caused by the ascomycete fungus Sclerotinia trifoliorum Erikss., is a major disease in Europe and reduces the persistence of red clover crops severely. The fungus infects clover plants through ascospores in the autumn, the disease develops during the winter and early spring and can kill many plants in this period. In early spring, black sclerotia, serving as surviving bodies, are formed on infected plants. Sclerotia can survive up to 7 years in the soil (Ohberg, 2006). The development of clover rot is highly dependent on the weather conditions: a humid fall, necessary for the germination of the ascospores and an overall warm winter with short periods of frost are favourable for the disease. Cold and dry winters slow the mycelial growth down too much and prevent the disease from spreading. Clover rot is difficult to control and completely resistant red clover varieties have yet to be developed. Because of the great annual variation in disease severity, plant breeders cannot use natural infection as an effective means to screen for resistant material. Breeding for resistant cultivars is being slowed down by the lack of a bio-test usable in breeding programs. When applying artificial infections, it is necessary to have an idea of the diversity of the pathogen. A diverse population will require resistance screening with multiple isolates. The objective of this research is to investigate the genetic diversity among isolates from the pathogen S. trifoliorum from various European countries. We assessed diversity using a species identification test based on the sequence of the beta-tubulin gene, vegetative compatibility grouping and AFLP.     

Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa

Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.     

OBSERVATIONS ON THE BIOLOGY OF CLOVER ROT

The field behaviour of clover rot in eastern England indicates that infection takes place by ascospores, and not by soil-borne mycelium developing directly from the sclerotia. To support this view, experimental evidence is presented to show that cutting of red clover at the time when the ascospores are being shed, reduces the percentage infection in the following spring.
Ascospore infection of clover causes a 'non-aggressive' spotting which may develop into an 'aggressive' infection. Sclerotinia trifoliorum has been shown to remain viable in non-aggressive spots for at least 16 weeks. Similar symptoms occur when beans are infected with S. trifoliorum var. fabae. Non-aggressive and aggressive infections are two phases of a sequence in time, and not two alternative types of infection dependent on the initial spore load. The occurrence of non-aggressive and aggressive phases in nature explains (1) the observed delay between ascospore discharge and the appearance of obvious disease symptoms, and (2) the seasonal nature of the disease.
Aggressive infection develops only in a saturated atmosphere and within a temperature range of 5–20° C., and its establishment is favoured by a heavy dosage of inoculum. In most seasons humidity is the main 'limiting' factor; over the 18-year period, 1930–47, there is a significant regression between the severity of clover rot and the previous December plus January rainfall.
Crop rotation and autumn grazing check the disease, but the latter must be practised with discretion because, under certain conditions, its effects are more drastic than those of the disease. The only hopeful method of effective control is the breeding of resistant strains of legumes.     

Leaf area index, canopy structure and photosynthesis of red clover (Trifolium pratense L.)

Abstract. The influence of leaf age, total leaf area and its dispersion in space on canopy photosynthesis were studied using microswards of red clover ( Trifolium pratense L.) which were established in the greenhouse. Two varieties, Renova (flowering) and Molstad (non-flowering), were sown in separate plastic boxes at densities of 225, 400 and 625 plants per m².
Vertical distribution of photosynthetically active radiation (PAR), leaf area, leaf age and ¹⁴CO₂-fixation were determined periodically. Net photosynthesis and dark respiration of canopies were measured. Maximum photosynthetic capacity of individual leaves was measured on plants taken from the intact canopy or from plants where shading of the growing leaves had been prevented.
Net photosynthetic rate of canopies increased linearly with leaf area index (LAI) up to an LAI of 3.5 and then declined at higher LAI, independent of variety and sowing density. Below the optimum LAI, net photosynthesis depended mainly on interception of PAR. Decrease in canopy photosynthesis above the optimum LAI was due to a higher proportion of old leaves with decreased photosynthetic capacity, and not to an increase in respiring plant parts. It is concluded that LAI and position of leaf age categories in the canopy are more important than vertical distribution of leaf area in determining canopy photosynthesis of red clover.     

Proteolysis and cell death in clover leaves is induced by grazing

Summary.  Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 °C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 °C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms. Received June 24, 2002; accepted August 15, 2002; published online March 11, 2003     

Susceptibility of clover species to fungal infection: the interaction of leaf surface traits and environment

Many foliar pathogens require free water to germinate; therefore, disease pressure should favor plants that are able to repel water. For a suite of 18 sympatric clover species (Trifolium and Medicago, Fabaceae), we evaluated leaf traits affecting leaf wetness and susceptibility to infection by the fungal pathogen Stemphylium sp., causal agent of Stemphylium leaf spot. Spore germination increased with time in free water, and the relative susceptibility of host plants to infection was proportional to the duration of water retention on leaves. Larger leaves captured more water and retained it longer. Unexpectedly, trichomes and leaf wettability did not affect water capture. For clovers planted within natural clover populations at two sites, infection was threefold greater at the wetter site. At the drier site, water retention on the leaf surface was an important predictor of infection rates across host species, but persistent fog and dew at the wetter site reduced the importance of rapid leaf drying. Our results suggest that plant adaptations that reduce water retention on leaves may also reduce disease incidence, but the selective advantage of these traits will vary among habitats.     

Measuring summer patterns of ascospore release by saltmarsh fungi

One potentially important type of flux from standing-decaying marshgrass is the production and release of ascospores. The most extensive measurements of ascospore release from the principal marshgrass (Spartina alterniflora, smooth cordgrass) of saltmarshes of the eastern coastal United States involved an arbitrary, weeklong period of wet incubation of leaf-blade samples. We examined the possibility that shorter incubations would yield higher estimates of hourly rates of ascospore release, testing wet incubations of 3 to 71 h, using standing-decaying leaf blades of smooth cordgrass from low on living shoots and high on dead shoots. Incubations of 31 h appeared to be optimal. Species compositions of ascospores expelled from the two leaf types were distinctly different: high leaves yielded primarily aMycosphaerella species orPhaeosphaeria halima; low leaves yielded primarilyPhaeosphaeria spartinicola or theMycosphaerella species. All of these species consistently exhibited high coefficients of variation (>100%) for their mean rates of release of ascospores. Only theMycosphaerella species on high leaves gave evidence of a delayed onset of ascospore expulsion during incubation, and this evidence was equivocal. Grand mean rates of ascospore release forP. spartinicola and theMycosphaerella species were, respectively, 106 and 238 spores cm⁻² abaxial leaf area h⁻¹.     

Genetic diversity analysis of isolates of the fungal bean pathogen Pseudocercospora griseola from central and southern Brazil

Planting resistant varieties is the most effective control measure against the angular leaf spot of dry beans, a fungal disease caused by Pseudocercospora griseola. However, dry bean varieties with durable resistance are not easily obtained. Knowledge about the genetic variability of the pathogen population is key for the success of dry bean breeding programs aimed at developing resistant materials, but finding suitable operationally simple and genetically accurate markers is not an easy task. The aim of this study was to assess the suitability of the ISSR-PCR technique to quantify the genetic variability of P. griseola isolates. Total DNA of 27 P. griseola isolates from Goiás, Minas Gerais, Espírito Santo, and Paraná States was extracted and amplified using specific primers for ISSR. Using cluster analysis, 27 genotypes were identified. The ISSR-PCR technique was suitable for assessing intraspecific variability of P. griseola. The ISSR-PCR marker was found to be highly sensitive to genetic variation and can aid in elucidating the genetic structure of the population of this plant pathogen as a support tool for the dry bean breeding programs.     

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.     

The effect of benomyl sprays on Sclerotinia trifoliorum and yield of red clover

Benomyl sprays, applied monthly from September to January, increased the survival and dry-matter yield of red clover and decreased the numbers of apothecia of Sclerotinia trifoliorum. Two sprays increased dry-matter yield in the first harvest year, if applied between October and December, but decreased yield in the second harvest year.     

Cutting reduces variation in biomass production of forage crops and allows low‐performers to catch up: A case study of Trifolium pratense L. (red clover)

• Re‐growth of fodder plants after grazing and mowing drives the profitability of their cultivation and is therefore an important target trait for plant breeding and agricultural engineering. However, for some fodder plants little is known about their re‐growth dynamics in response to grazing or mowing.
• We analysed the native response of plant architecture, leaf morphology and growth performance to experimental cutting in wild Trifolium pratense L. (red clover) plants. A total of 150 potted clover plants were established under controlled field conditions, and half of the plants were cut to 5 cm 3 months after sowing. Each plant was measured every week for 5 months.
• The cut and subsequently re‐grown plants carried fewer main branches (?20%), as well as fewer (?13%) and smaller (?32%) leaves than the control plants. However, the cut plants produced an average of 17% more accumulated leaf area (cut + re‐grown leaf area) than the control plants. This discrepancy was explained by variation in the growth strategy of the plants, where the cut plants invariably expressed a second growth phase, while almost half of the untreated plants did not.
• Our results suggest that cutting acted as an artificial trigger initiating a second growth phase in the cut plants and thereby contributed to yield increase. Exploiting this mechanism may set new goals for breeding and optimisation of the mowing regime.

     

THE CONFIRMATION OF THE VARIETY FABAE KEAY OF SCLEROTINIA TRIFOLIORUM ERIKSSON

There is confusion as to whether the Sclerotinia on Vicia faba is a normal strain of Sclerotinia trifoliorum as found on clover and related legumes or a distinct variety (var. fabae ). Pathologically the two fungi appear to be the same, and field observations indicate that beans in the rotation may aggravate the incidence of clover rot. A re-investigation of the morphology of the two fungi is made to assess the status of var. fabae.
The two fungi could not be distinguished by apothecial characters or cultural behaviour. Ascospore measurements confirm the existence of S. trifoliorum var. fabae , although 5. trifoliorum proper also occurs on beans. Var. fabae has not been found on any legume besides beans. Isolates from trefoil and sainfoin agree in spore size with S. trifoliorum on clover.
When isolates of S. trifoliorum are co-inoculated on the same Petri plate, they either do not react, or react to form a 'demarcation line'. Although a demarcation line is assumed to imply a genetic difference, non-reaction does not necessarily imply genetic identity. Non-reaction and the formation of a demarcation line also occur when var. fabae strains are co-inoculated together. When an isolate of S. trifoliorum is co-inoculated with an isolate of S. trifoliorum var. fabae an 'incompatibility line' is formed. Use can be made of this fact in identification.
On the basis of co-inoculations 5. trifoliorum and S. trifoliorum var. fabae are both shown to constitute a diverse range of biotypes. Segregation of biotypes in the ascus as a result of heterokaryosis in the ascus-initial is demonstrated. The view is expressed that heterokaryosis is the mechanism underlying the production of new biotypes.     

Nitrogen mineralization and crop uptake of N from decomposing 15N labelled red clover and yellow sweetclover plant fractions of different age

Nitrogen mineralization from 15N labelled red clover (Trifolium pratense L.) and yellow sweetclover (Melilotus officinalis Lam.) plant fractions of three different ages (8-, 14- and 20-week old) was studied in an out-door pot experiment during 8.5 months. Individual plant fractions (leaves/stems/roots/flowers), 23 g dry matter pot-1 (corresponding to 7300 kg ha-1), were incorporated into a sandy soil. The net mineralization of N was measured as 15N recovery in spring wheat (Triticum æstivum L.), perennial ryegrass (Lolium perenne L.) following the wheat and in the soil mineral N pool. Dry matter and N yields of the wheat crop were largest in pots receiving the legume leaf materials and the oldest root fractions. The largest amount of net N mineralized was obtained after application of sweetclover leaves, 381 mg N pot-1 (38% of added N), and a smaller amount was measured from red clover leaves, 215 mg N pot-1 (26% of added N). The N release was much smaller from the stems, being on average 63 mg N pot-1 (15% of added N), with intermediate values obtained from roots, 152 mg N pot-1 (26% of added N). The effects of age of the legume fractions on net N mineralization were more pronounced for sweetclover than for red clover materials. Greater net N release was obtained from sweetclover leaves and roots with inceasing age, but the opposite was valid for stems. At final harvest of the ryegrass, an additional 2.8% of added legume N was mineralized, compared with at wheat harvest. The net N mineralized proportion of added N was significantly related to concentrations of N and cell wall constituents in the plant material. Differences in net N mineralization estimates were generally larger between plant fractions than between materials of different age, implying that leaf proportion of the above-ground biomass is of great importance when predicting net N mineralization from green-manure plant materials. In addition, the contribution from roots to net mineralized legume N could be substantial.     

Infection of Subterranean Clover (Trifolium subterraneum) by Kabatiella caulivora

Kabatiella caulivora is a serious pathogen of clover ( Trifolium ) spp. Subterranean clover ( T. subterraneum ) cv. Woogenellup was inoculated with K. caulivora , to study the attachment and germination of conidia, germ-tube penetration of the plant surface, and histochemistry and ultrastructure of changes in the host associated with lesion development. The foliar architecture caused the conidia to concentrate at the base of leaflets and on the petiolules (between the leaflets and petioles). Epidermal cells immediately beneath conidia and, occasionally, also adjacent cells developed a yellow-brown discoloration 1 day post-inoculation. Penetration appeared to be directly through the cuticle, characterized by constricted hyphae at the point of entry. No appressoria were observed. In leaves, invasion was restricted to the area proximal to the petiolule and leaf mid-rib. In petioles and petiolules, the hyphae initially remained between the epidermal cells and first layer of mesophyll cells before moving intercellularly through the mesophyll tissue towards phloem tissues. The cuticle was occasionally degraded in petiole and petiolule infections, the loss of epidermal and mesophyll cell wall components was detected, and chloroplasts and starch grains were disrupted. Plants developed macroscopic symptoms 10–11 days post-inoculation with necrotic lesions occurring on leaves, petioles and petiolules. Sporulation occurred approximately 15–18 days post-inoculation when affected plants collapsed. This information may be useful for breeding programmes aimed at selecting varieties with improved resistance to the clover scorch disease.     

The Ecology of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> on the Phylloplane: Colonization from Soil,Plasmid Transfer,and Interaction with Larvae of <Emphasis Type="Italic">Pieris brassicae</Emphasis>

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.     

Patterns of 14C-labelled Assimilate Partitioning in Red and White Clover During Vegetative Growth

A quantitative analysis of the ¹⁴C-labelled assimilate suppliedby the expanded leaves on the primary shoot to growing leaves,stem, lateral shoots (branches or stolons) and roots in redand white clover was conducted during vegetative growth. Stem growth of the primary shoot was inhibited in both cloversand utilized no energy resources. The growing leaves at theprimary shoot apex of white clover imported 4 per cent of theshoot's assimilate compared with 10 per cent in red clover.At the basal end of the primary shoot, the tap root of whiteclover imported 16 per cent of the shoot's assimilate comparedwith 22 per cent in red clover. Branches in red clover and stolonsin white clover were by far the largest sinks for primary shootassimilate, importing 39 per cent and 63 per cent of the labelledassimilate, respectively. Analyses of the translocation of assimilate from individualprimary shoot leaves demonstrated that in both clovers olderleaves exported more of their assimilate to branches or stolons,whereas younger leaves exported more of their assimilate toroots, and possibly in white clover, to growing leaves at thetip of the shoot. Of the labelled assimilate exported to branchesor stolons, each primary shoot leaf exported preferentiallyto the branch or stolon in its own axil, but in addition exportedsubstantial quantities of assimilate to all other axillary shoots,particularly those arising from basal axils where the subtendingleaf had died. Trifolium repens, Trifolium pratense, red clover, white clover, assimilate partitioning, perennation     

The elimination of clover diseases by shoot tip culture

Shoot tip culture was used to eliminate white clover mosaic virus (WCMV) and red clover necrotic mosaic virus (RCNMV) from red clover, and clover phyllody disease (CP) and clover red leaf disease (CRL) from white clover. Shoot tips up to 2.4 mm (in some cases 3 mm) could regenerate plants free from the pathogens, but the efficiency of elimination, at least for WCMV and CRL, tended to decrease with increasing shoot tip size. The efficiency of plant regeneration from shoot tips generally improved with increasing tip size.     

Feeding site selection of first-instar larvae of Cnaphalocrocis medinalis on susceptible and resistant rice plants

More first-instar larvae of the rice leaffolder, Cnaphalocrocis medinalis (Guenée), were recovered from susceptible IR36 and Roxoro rice varieties than resistant TKM6 and Oryza perennis, 24 h after infestation. Within a rice plant, a higher proportion of larvae was recovered from young whorl leaf, followed by mature leaves and leafsheaths on all the four rice varieties tested. However, differences were observed between susceptible and resistant varieties in the distribution of larvae on these three plant parts. In laboratory choice tests, first-instar larvae preferred to settle on the young leaves of IR36 and TKM6 when presented with their respective mature leaves. No such preference was observed in tests with Rexoro and O. perennis. Larval survival was similar on young and mature leaves of Rexoro. Young leaves of IR36 and TKM6 were more suitable for survival in comparison with their respective mature leaves while the reverse was true for O. perennis. Larval movement was slower on the mature leaves and larvae took longer to reach the whorl leaf of TKM6 than on IR36. The density of trichomes of the abaxial surface of TKM6 was higher than that of the other varieties tested. Adaptive significance of feeding in the leafwhorl to young larvae is discussed.     

Intracellular Localization of Two Enzymes Involved in Coumarin Biosynthesis in Melilotus alba

The localization of phenylalanine ammonia-lyase [EC 4.3.1.5] within sweet clover (Melilotus alba) leaves was investigated. Apical buds and axillary leaves contained 15 to 30 times more enzyme activity than did mature leaves. Mesophyll protoplasts were prepared by digesting young leaves with Cellulysin and Macerase and were gently ruptured yielding intact chloroplasts. These chloroplast preparations exhibited neither phenylalanine ammonia-lyase nor o-coumaric acid O-glucosyltransferase activities. The general enzymic properties of sweet clover leaf phenylalanine ammonia-lyase were similar to those described for this enzyme isolated from other plant species. The conversion of l-phenylalanine to trans-cinnamic acid, which occurred at an optimum pH of about 8.7, was strongly inhibited by the metabolites trans-cinnamic and o-coumaric acids. In contrast, o-coumaric acid glucoside, coumarin, p-coumaric acid, and melilotic acid had no significant effect on the reaction rate.