Reduction of sclerotial inoculum of Sclerotinia sclerotiorum with Coniothyrium minitans

Aspects of the biology of C. minitans and its potential for control of S. sclerotiorum were investigated.Temperatures below 7°C resulted in comparatively slow rates of germination and infection of sclerotia by C. minitans. The optimum temperature for germination, growth, infection of sclerotia, and destructive parasitism by C. minitans was 20°C. The optimum relative humidity for germination, growth and infection by C. minitans was above 95%.Autumn inoculations with suspensions of conidia, pycnidia and mycelium of C. minitans in the field resulted in negligible numbers of sclerotia remaining viable after 1 month. With culture-grown sclerotia 2 months were required for a similar reduction of sclerotial viability. In the absence of C. minitans mulching had no significant effect on sclerotial viability. In the presence of C. minitans mulching did, however, influence the viability and infection by C. minitans of culture-grown sclerotia. Populations of field sclerotia also differed from culture-grown sclerotia in that they harboured an internal population of microorganisms, which included C. minitans, and had a lower level of viability at the commencement of the treatments.A winter application of C. minitans did not result in significant infection of sclerotia nor in a reduction in viability of sclerotia. This failure is believed to have resulted from low temperatures and dry conditions.     

Evaluation of different Coniothyrium minitans inoculum sources and application rates on apothecial production and infection of Sclerotinia sclerotiorum sclerotia

The effects of three Coniothyrium minitans isolates (Conio, IVT1 and Contans^®), applied to soil as conidial suspensions or as maizemeal-perlite (MP) inocula (Conio), on apothecial production and infection of Sclerotinia sclerotiorum sclerotia were assessed in two soil pot bioassays and two novel box bioassays in the glasshouse at different times of the year. C. minitans isolate Conio applied as either MP or ground MP at full rate (10⁶-10⁷ cfu cm⁻³ soil) consistently decreased the carpogenic germination, recovery and viability of sclerotia and increased C. minitans infection of the sclerotia of S. sclerotiorum by in comparison with either MP or conidial suspension treatments applied at lower rates (10³-10⁴ cfu cm⁻³ soil). Additionally, when applied at the same rate, MP inoculum of C. minitans was consistently more effective at reducing carpogenic germination than a conidial suspension. The effect of MP and ground MP at full rate on carpogenic germination was expressed relatively early as those sclerotia recovered before apothecia appeared on the soil surface already had reduced numbers of apothecial initials. In general, there were few differences between the isolates of C. minitans applied as conidial suspensions. Box bioassays carried out at different times of the year indicated that temperature and soil moisture influenced both apothecial production and mycoparasitism. Inoculum concentration of C. minitans and time of application appear to be important factors in reducting apothecial production by S. sclerotiorum.     

Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils

The biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 were added separately to three soil types that had been either sterilised, pasteurised or left non-sterile. Applied as a conidial suspension of 1×10⁶ cfu g⁻¹ soil, C. minitans showed good survival in all sterilised, pasteurised and non-sterile soils, remaining at the numerical level at which it was applied for the duration of the 30 d experiment. Applied at a lower rate of 1×10³ cfu g⁻¹ soil, C. minitans proliferated in sterilised soil to numbers slightly over 1×10⁶ cfu g⁻¹ soil, whereas no increase was seen in pasteurised or non-sterile soils from this lower application rate. However, although C. minitans was not easily recovered on plates from non-sterile soil, it did survive at the lower numerical level in pasteurised soil, and was recoverable throughout the experiment at the rate at which it was applied. B. subtilis MBI 600 survived well following introduction as a cell suspension into sterilised soil at a rate of 1×10⁶ cfu g⁻¹ soil. Spores were formed rapidly and, after 14 d, the introduced microorganism survived in this form rather than as vegetative cells. However, in non-sterile soil, the introduced microorganism did not compete well and decreased in number, with spores being formed in low numbers. Survival of B. subtilis MBI 600 in pasteurised soil was variable, but resembled the survival seen in non-sterile soil more than that seen in sterilised soil. More B. subtilis MBI 600 spores were formed in pasteurised soil than in non-sterile soil, however, and may have been important for survival in pasteurised soil. In conclusion, this work has shown that the biocontrol agent C. minitans can survive well in soil irrespective of whether the soil has been pasteurised or not and shows good promise as a soil inoculant for control of Sclerotinia sclerotiorum. Although soil pasteurisation does improve establishment of B. subtilis MBI 600 compared to non-sterile soil, survival is relatively poor when applied as cells. The best survival of B. subtilis MBI 600 occurred as spores in sterilised soil, and spore applications to pasteurised soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off.     

Time and burial depth influencing the viability and bacterial colonization of sclerotia of Sclerotinia sclerotiorum

Sclerotia are the primary over wintering inoculum of Sclerotinia sclerotiorum (Lib.) de Bary. The effects of tillage on the primary inoculum are not well understood. The purpose of this research was to study sclerotial viability over time and between burial depths in soil, to identify bacteria colonizing and degrading the sclerotia, and determine whether these bacteria may be utilized as biological control agents. Correlation analysis indicated that a significant negative relationship existed between sclerotial viability and elapsed temporal factors (R²=−0.68, P<0.0001), and depth of burial (R²=−0.58, P<0.0001). After twelve months, sclerotia on the soil surface had the highest viability (57.5%), followed by those at the 5 cm depth (12.5%), and only 2.5% of those placed at the 10 cm depth remained viable. A significant negative relationship between sclerotial viability and bacterial populations also existed (R²=−0.60, P<0.0001). Two hundred and sixty-eight bacteria were isolated from sclerotia, 29 of which showed strong in vitro antagonism to the mycelial growth of S. sclerotiorum. Biodiversity of the inhibitory bacterial isolates was minimal on sclerotia from the soil surface and within all depths sampled at three months (i.e. in January). All burial depths within the April and July sampling dates produced bacterial diversities that were distinct from each other.     

Parasitism of sclerotia of Sclerotium rolfsii by trichoderma harzianum

The ability of Trichoderma harzianum isolate 203 to attack the soil-borne plant pathogen Sclerotium rolfsii is apparently connected with the production by the isolates of chitinase and β-(1,3)-glucanase inside the attacked sclerotia during parasitism.SEM and TEM micrographs show that the mycoparasite degraded walls of sclerotial cells and the attacked cells lost their cytoplasmic contents. It is assumed that T. harzianum utilizes sclerotial cell contents thus enabling it to sporulate intensively on the sclerotial surface and inside the digested cells.     

Energy efficiency of the mycoparasite Sporidesmium sclerotivorum in vitro and in soil

The energy content of the mycoparasite Sporidesmium sclerotivorum mycelium was 18,389 J g^?1 and 16,334 J g^?1 for macroconidia on a dry weight basis. The energy content of Sclerotinia minor sclerotia, the host of the mycoparasite, was 16,485 J g^?1. In liquid culture, the economic coefficient for the conversion of glucose to mycelium (mycelial dry wt ÷ glucose consumed × 100) was 51–60 whereas the mycelial energy coefficient, [mycelial energy (J) ÷ substrate energy (J) × 100] was 65–75. In soil, the conidial energy coefficient [conidial energy (J) ÷ substrate energy (J) × 100] for the conversion of host sclerotial energy to the macroconidia of the mycoparasite was 19.8, which was 2–9 times that for the conversion of glucose in liquid culture. The conidial energy coefficient when grown on a liquid medium on vermiculite was 23.0. S. sclerotivorum, as an obligate parasite of sclerotia in soil, was most efficient in the conversion of energy in a system where there was a high surface: energy ratio. In liquid culture S. sclerotivorum is more efficient than most other fungi.     

Comparaison des aptitudes parasitaires de clones de Trichoderma vis-a-vis de quelques champignons a sclerotes

Nineteen monoconidial isolates (referred to as clones) of Trichoderma from different species aggregates, one isolate of Gliocladium virens, and one isolate of an Acrostalagmus sp. (that was naturally associated with sclerotia of Sclerotinia spp and Macrophomina phaseolina) were tested. They were incubated in controlled conditions, in sterile soil, with sclerotia of Corticium rolfsii, Sclerotinia minor, or S. sclerotiorum. At the end of appropriate periods of incubation (respectively 26, 20 and 8 days), the sclerotia were retrieved from soil and checked for invasion by the antagonist. Important differences between the parasitic ability of Trichoderma clones were noted. Clones from at least three different species (T. aureoviride, T. hamatum, T. harzianum) exhibited a high antagonistic activity. Activity of the G. virens isolate was at the same level as the best clones of Trichoderma, whereas no parasitic tendencies were found in the isolate of Acrostalagmus sp., thus confirming previous results.A rather good correlation was found between the capacity of the clones for attacking C. rolfsii sclerotia and their ability to parasitize both Sclerotinia.In conclusion, it is proposed that a screening with only one of the sclerotial species would give clones efficient against all three, and possibly against related sclerotial types.     

Microbial populations involved in the suppression of Rhizoctonia solani AG1-1B by lignin incorporation in soil

Rhizoctonia solani causes worldwide losses in numerous crops. Sclerotia of R. solani remain viable for several years in soil and are an important source of primary infection. In this study the effect of soil incorporation of Kraft pine lignin, a side product of the paper industry, on viability of R. solani AG1-1B sclerotia was investigated. The efficacy of lignin was assessed in a sandy loam (Oppuurs) and a silt loam soil (Leest) collected from commercial fields in Belgium. Evaluating sclerotial viability after 4 weeks incubation in the two soils amended with 1% (w/w) Kraft pine lignin demonstrated a soil-dependent effect. In Leest soil the addition of lignin resulted in a significantly reduced sclerotial viability, together with an increased mycoparasitism by Trichoderma spp.; in Oppuurs soil, on the other hand, only a slight and insignificant reduction in sclerotial viability was observed. Based on phospholipid fatty acid analysis, different changes in microbial community structure upon lignin amendment were detected in the two soils. Both amended soils showed a significant increase in Gram negative bacteria. In Leest soil this increase was accompanied with a significantly higher increase in fungi and actinomycetes compared with Oppuurs soil. In addition, Kraft pine lignin resulted in both soils in a small but significant increase in manganese peroxidase activity and this increase tended to be higher in Leest soil. Manganese peroxidase produced by lignin-degrading basidiomycetes has previously been shown to degrade melanin, which protects the sclerotia against biotic and abiotic stress. We hypothesize that lignin-degrading fungi increased the susceptibility of the sclerotia to sclerotial antagonists such as Trichoderma, Gram negative bacteria and actinomycetes. Clearly, the effect observed here did not rely on the stimulation of one microbial group, but is the result of an interaction of different groups.     

Survival of sclerotia of Macrophomina phaseolina and Sclerotium cepivorum after drying and wetting treatments

Exposure of sclerotia of Macrophomina phaseolina to 0 and 33% relative humidity (r.h.) for 12 weeks and of Sclerotium cepivorum to 0, 33 and 55% r.h. for 20 weeks did not reduce their germinability on agar. Exposure to 78% r.h. caused high loss of germinability in M. phaseolina and complete loss in S. cepivorum. After 7-day exposures respective moisture contents of sclerotia of M. phaseolina and S. cepivorum were 1 and 2% at 0% r.h.; and 10 and 14% at 78% r.h. M. phaseolina sclerotia held at 0% and 33% r.h. in desiccators for several times up to 12 days did not decrease in subsequent survivability in moist soil, unlike sclerotia held at 78% r.h. for 4 days.More sclerotia of M. phaseolina were colonized by fungi and Streptomyces spp. on alkaline soil than on acid soil. On alkaline soil twice as many sclerotia were colonized after exposure to 0% r.h. as after exposure to 33, 55 and 78% r.h. Colonization of S. cepivorum sclerotia was as high on acid as on alkaline soil and 3 times as high on sclerotia treated at 0% r.h. as on those treated at higher r.h. Attempts to ascertain the effects of colonization on sclerotial viability were unsuccessful. Incubation of sclerotia of M. phaseolina in moist Rumsford sandy loam (50% m.h.c.) for 20 weeks reduced survivability by 43%. At room temperature, alternate drying and wetting of soil containing sclerotia did not appreciably affect survivability of either pathogen. Survivability of S. cepivorum sclerotia was highest when the sclerotia were incubated in air-dried soil (2–3% m.h.c.) for 20 weeks.Incidence of white rot on onion seedlings transplanted to S. cepivorum-infested soil was higher in soil that had been air-dried for 20 weeks than in soil that had been alternately wetted and dried. Sclerotia that were exposed to 0% r.h. for 7 days before soil incubation produced little white rot.     

Population dynamics of the mycoparasite, sporidesmium sclerotivorum, and its host, sclerotinia minor, in soil

The infection and survival of sclerotia of Sclerotinia minor and the production ofmacroconidia of the mycoparasite, Sporidesmium sclerotivorum, were studied in vitro when each fungus was added to soil at various initial inoculum densities. The rate at which S. sclerotivorum invaded host sclerotia and caused their decay varied with the amount of the mycoparasite added to soil. The results suggest that approximately 5 macroconidia of the mycoparasite g^?1 of soil are needed to successfully infect sclerotia and bring about their decay, when soils are sampled and mixed every 2 weeks. The rate at which S. sclerotivorum infects sclerotia of S. minor and causes their decay is also dependent on the initial inoculum density of the host. Each infected sclerotium supports the production of about 15,000 new macroconidia in soil regardless of the initial inoculum density of the host. It is concluded that successful biological control by S. sclerotivorum is dependent on the soil population of both the host and the mycoparasite.     

Interactions between Sclerotium rolfsii and Trichoderma spp: Relationship between antagonism and disease control

Ten isolates of Trichoderma spp were examined for their ability to antagonize growth and to parasitize mycelium of Sclerotium rolfsii (Sr-1) on agar media, to inhibit germination of sclerotia of S. rolfsii on natural soil plates and to sporulate on the sclerotia, and to protect bean seedlings against the pathogen in the greenhouse. A high negative correlation (r = ?0.844) was observed between plant stand in the greenhouse and sclerotial germination on soil plates but not with antagonism on agar plates. Three isolates of T. harzianum (Th-7, Th-20, WT-6) and one of T. hamatum (TRI-4) were especially effective in reducing sclerotial germination and controlling disease in the greenhouse. Three isolates of Trichoderma spp (WT-6, TMP, and TRI-4), effective in reducing sclerotial germination of isolate Sr-1, also prevented sclerotial germination in four out of five additional S. rolfsii isolates studied.     

Identification and use of potential bacterial organic antifungal volatiles in biocontrol

Bacteria, isolated from canola and soybean plants, produced antifungal organic volatile compounds. These compounds inhibited sclerotia and ascospore germination, and mycelial growth of Sclerotinia sclerotiorum, in vitro and in soil tests. Ascospore germination in cavity slides was inhibited 54-90% by the volatile producers. When mycelial plugs or the sclerotia, exposed to these volatiles, were transferred to fresh agar plates, the pathogen could not grow, indicating the fungicidal nature of the volatiles. Head space volatiles, produced by bacteria, were trapped with activated charcoal, by passing nitrogen continuously over shake cultures for 48 h. The compounds were eluted from the charcoal with methylene chloride and identified using Gas Chromatography-Mass Spectrometry (GC-MS). The volatile compounds included aldehydes, alcohols, ketones and sulfides. Of the 23 compounds assayed for antifungal activity in divided Petri plates, with filter-disks soaked with these compounds (100 and 150 μl), only six compounds completely inhibited mycelial growth or sclerotia formation, suggesting their potential role in biological control. The compounds are benzothiazole, cyclohexanol, n-decanal, dimethyl trisulfide, 2-ethyl 1-hexanol, and nonanal. Volatiles may play an important role in the inhibition of sclerotial activity, limiting ascospore production, and reducing disease levels. Studies are under way to understand this phenomenon under field conditions. This is the first report on the identification and use of bacterial antifungal organic volatiles in biocontrol.     

Diallyl-disulphide to reduce the numbers of sclerotia of Sclerotium cepivorum in soil

A single injection of 0.2 ml diallyl disulphide (DADS) at 0.156% (v/v) into soil containing naturally-produced sclerotia of Sclerotium cepivorum and maintained in the laboratory at 15°C stimulated sclerotial germination and reduced sclerotial numbers by 67%; ungerminated sclerotia remained viable. Higher concentrations of DADS had no additional effect except that at 20% (v/v), germination was slightly inhibited. A similar reduction in sclerotial numbers was obtained when the mixture of soil and sclerotia was exposed to DADS vapour. Four, monthly applications of DADS at 0.2 ml 0.15% (v/v) per application did not give a further reduction.The effect of DADS was temperature dependant, with a reduction in sclerotial numbers of 65 and 9% at 15 and 5°C respectively.     

The effect of dry conditions on subsequent leakage and rotting of fungal sclerotia

Dried sclerotia of Sclerotium delphinii rotted in moist soil whereas those of Sclerotium cepivorum. Botrytis cinerea and Botrytis tulipae did not. A number of fungi invaded dried sclerotia of S. delphinii in soil, the principal coloniser found in the first sampling being Trichodermu hamatum. Leakage of ¹⁴C compounds from dried labelled sclerotia placed in water was rapid and was little affected by variation in leakage temperature from 1 to 25°C or by prolonging the drying period beyond a day. Leakage from dried sclerotia which were allowed to imbibe water through a small part of their surface was much reduced. Sclerotia which were redried after leakage leaked again when returned to water but with all four fungi the first of three leakage cycles gave the highest ¹⁴C levels. Loss in dry weight in the first leakage cycle was greater with S. delphinii than with the other three fungi and this may be related to the poor survival of dried sclerotia of S. delphinii in moist soil. Substances lost during leakage appear to originate from within sclerotial hyphae rather than from the hyphal free space.     

Diversity of potential microbial parasites colonizing sclerotia of Macrophomina phaseolina in soil

The colonization of Macrophomina phaseolina sclerotia by microbial parasites was evaluated in unsterilized field soil at different levels of soil moisture (0,-5, and-10 kPa) and temperature (20, 30, and 40°C). The maximum colonization of sclerotia was recorded in soil held at-5 or-10 kPa at 30–40°C. Trichoderma harzianum isolate 25–92 and Pseudomonas fluorescens isolate 4–92 were recorded as potential sclerotial parasites, and they significantly (P=0.05) reduced the germination of sclerotia by 60–63%. Cells of P. fluorescens and buffer-washed conidia of T. harzianum were completely agglutinated at 28°C with crude agglutinin of M. phaseolina. The ability of different antagonists to parasitize the sclerotia were correlated with the agglutination ability of the antagonists.     

Quantitative real-time PCR effectively detects and quantifies colonization of sclerotia of Sclerotinia sclerotiorum by Trichoderma spp.

Some members of the fungal genus Trichoderma are able to colonize and destroy sclerotia, the thick-walled resting structures of the soilborne plant pathogenic fungus Sclerotinia sclerotiorum, thus providing a potential means of biological disease control. However, current methods to detect and quantify colonization of sclerotia are labor-intensive, and generally qualitative rather than quantitative in nature. Our objective was to develop quantitative real-time PCR (polymerase chain reaction) methods to detect and measure colonization of sclerotia by Trichoderma spp. Specific PCR primer/probe sets were developed for Trichoderma spp. and for S. sclerotiorum. A total of 180 ITS1 (internal transcribed spacer) and ITS2 sequences from different species in the genus Trichoderma were aligned, and consensus sequences were determined. Six candidate primer sets were based on conserved regions of the consensus sequence, and the specificity of each nucleotide sequence was examined using BLAST (Basic Local Alignment Search Tool; NCBI) software. Each candidate primer set was tested on genomic DNA of T. harzianum strain ThzID1-M3, as well as six different Trichoderma isolates from soil, and on genomic DNA of S. sclerotiorum. The optimum primer/probe set selected, TGP4, successfully amplified genomic DNA of all Trichoderma isolates tested, and showed high precision and reproducibility over a linear range of eight orders of magnitude, from 85 ng to 8.5 fg of T. harzianum genomic DNA. TGP4 did not amplify S. sclerotiorum DNA. A specific PCR primer/probe set (TMSCL2) was developed for S. sclerotiorum, based on the calmodulin gene sequence. TMSCL2 did not amplify Trichoderma DNA. Quantitative real-time PCR with the primers then was evaluated in experiments to test differential effects of two soil moisture levels (−50 kPa, −1000 kPa matric potential) on biocontrol of S. sclerotiorum by indigenous Trichoderma spp. Periodically over 40 days, Trichoderma and S. sclerotiorum DNA in sclerotia were quantified by PCR with appropriate primers. Over 90% of the sclerotia were colonized by indigenous Trichoderma spp. at −1000 kPa, over the 40-day period, compared to only 60% at −50 kPa. In addition to determining incidence of colonization, the PCR method allowed measurement of the extent of sclerotial colonization, which also was significantly greater in the drier soil. Quantitative real-time PCR with the TGP4 primer/probe set provides a sensitive detection and measurement tool to evaluate colonization of sclerotia by Trichoderma spp.     

Control of apothecia of Sclerotinia sclerotiorum by soil amendment with S-H mixture or Perlka in bean, canola and wheat fields

Ascospores of Sclerotinia sclerotiorum produced from apothecia are the primary source of inoculum for causing diseases such as white mold of common bean, pod rot of pea, stem blight of canola and head rot of sunflower and safflower in the Canadian prairies. A field study was conducted for 4 years to determine efficacy of control of production of apothecia from carpogenically germinated sclerotia of S. sclerotiorum by soil amendment with Perlka^® (calcium cyanamide) and S-H mixture (a formulated compound). Results of the 4-year experiments showed that amendment of soil with Perlka^® at low (30 g/m²) or high (60 g/m²) rate was effective in reducing carpogenic germination of sclerotia and production of apothecia under the canopy of host crops (common bean and canola) and a non-host crop (wheat). In the experiments of 1988, for example, the numbers of apothecia produced in the treatments of Perlka^®-low rate (30 g/m²), Perlka^®-high rate (60 g/m²) and untreated control were 42, 46, and 182 apothecia/plot (m²), respectively, for bean; 89, 42, and 318 apothecia/plot (m²), respectively, for canola; and 146, 143, and 412 apothecia/plot (m²), respectively, for wheat. However, soil amendment of S-H mixture at low (30 g/m²) or high (60 g/m²) rate was ineffective in reducing carpogenic germination of sclerotia and production of apothecia for all the 4 years of testing in all three crops. The ineffectiveness of S-H mixture and the practicality of Perlka^® for control of Sclerotinia diseases of crops grown under Canadian prairie conditions are discussed.     

Potential gene exchange between Bacillus thuringiensis subsp. kurstaki and Bacillus spp. in soil in situ

The possible transfer of genes from Bacillus thuringiensis subsp. kurstaki (Btk) to indigenous Bacillus spp. was investigated in soil samples from stands of cork oak in Orotelli (Sardinia, Italy) collected 5 years after spraying of the stands with a commercial insecticidal preparation (FORAY 48B) of Btk. Two colonies with a morphology different from that of Btk were isolated and identified as Bacillus mycoides by morphological and physiological characteristics and by 16S rDNA analysis. Amplification by the polymerase chain reaction (PCR) of the DNA of the two isolated B. mycoides colonies with primers used for the identification of the Btk cry genes showed the presence of a fragment of 238 bp of the cry1Ab9 gene that had a similarity of 100% with the sequence of the cry1Ab9 gene present in GenBank, indicating that the isolates of B. mycoides acquired part of the sequence of this gene from Btk. No cells of Btk or B. mycoides carrying the 238-bp fragment of the cry1Ab9 gene were isolated from samples of unsprayed control soil. However, the isolates of B. mycoides were not able to express the partial Cry1Ab protein. Hybridization with probes for IS231 and the cry1Ab9 gene suggested that the inverted repeated sequence, IS231, was probably involved in the transfer of the 238-bp fragment from Btk to B. mycoides. These results indicate that transfer of genes between introduced Btk and indigenous Bacillus spp. can occur in soil under field conditions.     

Molecular markers and a sequence deletion in intron 2 of the putative partial homologue of LEAFY reveal geographical structure to genetic diversity in the acutely threatened legume genus Clianthus

Clianthus is an acutely threatened, bird-pollinated genus endemic to New Zealand, represented in the wild by only one population of C. puniceus and 11 populations of C. maximus, each with very few individuals (typically <10 per population). A limited number of named Clianthus cultivars of indeterminate origin are commonly grown as ornamentals. Genomic DNA from individual Clianthus plants was extracted for genetic diversity analysis using a range of molecular markers, including amplified fragment length polymorphism (AFLP). Data were analysed by the unweighted pair-group method with arithmetic averaging (UPGMA), the generation of Neighbor-Joining trees, and analyses of molecular variance (AMOVA). Genetic distance between wild populations of C. maximus was highly correlated with geographical distance between populations. Sequencing of intron 2 of a putative partial homologue of the floral meristem identity gene LEAFY (CmLFY) revealed a 7 bp deletion that was exhibited homozygously in the more northern populations of C. maximus, and in all individuals tested from the sole population of C. puniceus. This deletion was not exhibited in more southern populations of C. maximus. Further, one geographically intermediate population contained some plants that were heterozygous for the deletion. Parallel analyses of cultivated Clianthus genotypes, more than half of which were also homozygous for the 7 bp deletion, showed that these were not representative of the broad, but threatened, diversity remaining in the wild. It is argued that wild populations of C. maximus are unlikely to have arisen from the escape of plants from cultivation. Conservation effort should focus on the protection and study of the extant plants in these wild populations, rather than on the introduction of disturbance regimes to uncover potential seed banks.     

Survival of sclerotia of sclerotinia sclerotiorum in soil

Factors affecting longevity of sclerotia in sandy clay loam (s.c.l.) and sandy loam (s.l.) were examined, using sclerotia from a laboratory culture of S. sclerotiorum and from natural infestations on beans and lettuce.Survival of sclerotia from culture and lettuce was compared in s.l. Recovery and viability were less, and incidence of Fusarium, Mucor and Trichoderma spp. greater, in sclerotia from lettuce than from culture. Rinds of sclerotia from lettuce were more perforated than those from culture.Burial of sclerotia at 4 cm for 35 weeks reduced recovery of sclerotia to zero in s.c.l. and by 50% in s.l. At the soil surface recovery was reduced by 55% in s.c.l. and by 10% in s.l. Less than 50% of sclerotia recovered were viable. Neither a chloropicrin-methyl bromide fumigant nor a tomato compost treatment affected recovery or viability. Fumigation increased incidence of Trichoderma spp. and decreased incidence of Fusurium and Mucor spp. isolated from sclerotia.Apothecia were produced over 6 weeks in s.c.l. and over 20 weeks in s.l. Production was increased by the low rate of fumigant in s.c.l. and by tomato compost in s.l.