Interactions of two selected field isolates of Colletotrichum gloeosporioides f. sp. aeschynomene on Aeschynomene virginica

The fungal plant pathogen, Colletotrichum gloeosporioides f. sp. aeschynomene (CGA), has been used successfully since 1982 to control northern jointvetch (NJV), Aeschynomene virginica, in rice and soybeans in the USA as COLLEGO^®, a bioherbicidal formulation of a single isolate of this pathogen. The interactions and fitness of different field isolates of this fungus and effective bioherbicide have not been fully examined. We examined CGA population structure and inter-isolate competition using molecular markers in field and greenhouse experiments. NJV plants were first inoculated with one isolate or a mixture of two isolates, Cla-5a and 3-1-3, followed by a challenge inoculation containing either one or both isolates, 4 or 5 days later. Plants first inoculated with Cla-5a alone, or in mixture with 3-1-3, had Cla-5a comprising approximately 80% of the population after 21 days regardless of challenge inoculation. In contrast, when the primary treatment was 3-1-3 alone, wounding, or benzodiathiazole, 3-1-3 detection only approached 50% of the population following challenge inoculation. Primary inoculation with Cla-5a significantly reduced the number of lesions caused by 3-1-3 challenge, whereas primary inoculation with 3-1-3 did not significantly affect the number of lesions caused by Cla-5a challenge. Size of lesions caused by the two isolates following challenge inoculations and dispersal of the isolates from lesions to healthy plants were not affected by the primary inoculation. These results suggest a specific interaction by Cla-5a with the host defense response creating a more favorable environment for its own colonization of NJV to the exclusion of 3-1-3.     

Soybean and sunflower oils increase the infectivity of Colletotrichum gloeosporioides f. sp. aeschynomene to Northern Jointvetch

Soybean and sunflower oils increased the level of infection of northern jointvetch, Aeschynomene virginica, plants by Colletotrichum gloeosporioides f. sp. aeschynomene. Inoculation of seedlings with spore suspensions containing 10% (v:v) soybean oil or 10% sunflower oil resulted in more disease than when inoculated with suspensions of spores in water alone. The lengths of the dew periods required to establish equivalent levels of disease by spore suspensions containing 10% soybean or 10% sunflower oil were approximately 4–8 h less compared to aqueous suspensions. Incubation of spores in 10% soybean oil followed by removal and resuspension in water did not affect the infectivity of spores when compared to spores incubated in aqueous suspensions. Spore germination and appressoria formation were unaffected by either of the oils tested in in vitro assays; however, in in vivo assays, 10% soybean oil and 10% sunflower oil increased spore germination in comparison to spores that were suspended in water.     

Best linear unbiased prediction of host-range of the facultative parasite Colletotrichum gloeosporioides f. sp. salsolae, a potential biological control agent of Russian thistle

Russian thistle or tumbleweed (Salsola tragus L.) is an introduced invasive weed in N. America. It is widely distributed in the US and is a target of biological control efforts.The fungus Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in Penz. f. sp. salsolae (CGS) is a facultative parasite under evaluation for classical biological control of this weed. Host-range tests were conducted with CGS in quarantine to determine whether the fungus is safe to release in N. America. Ninetytwo accessions were analyzed from 19 families: Aizoaceae, Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Campanulaceae, Chenopodiaceae, Cucurbitaceae, Cupressaceae, Fabaceae, Malvaceae, Nyctaginaceae, Phytolaccaceae, Poaceae, Polygonaceae, Sarcobataceae, and Solanaceae and 10 tribes within the Chenopodiaceae: Atripliceae, Beteae, Camphorosmeae, Chenopodieae, Corispermeae, Halopepideae, Polycnemeae, Salicornieae, Salsoleae, and Suaedeae. These included 62 genera and 120 species. To facilitate interpretation of results, disease reaction data were combined with a relationship matrix derived from internal transcribed spacer DNA sequences and analyzed with mixed model equations to produce Best Linear Unbiased Predictors (BLUPs) for each species. Twenty-nine species (30 accessions) from seven closely-related Chenopodiaceae tribes had significant levels of disease severity as indicated by BLUPs, compared to six species determined to be susceptible with least squares means estimates. The 29 susceptible species were: 1 from Atripliceae, 4 from Camphorosmeae, 1 from Halopepideae, 2 from Polycnemeae, 6 from Salicornieae, 8 from Salsolae, and 7 from Suaedeae. Most species in the genus Salsola, which are all introduced and weedy, were very susceptible and damaged by CGS. Statistical comparisons and contrasts of BLUPs indicated that these Salsola species were significantly more susceptible than non-target species, including 15 species from relatives in the closely-related genera Bassia (=Kochia), Nitrophila, Salicornia, Sarcocornia, and Suaeda. Of the 29 susceptible species, 10 native or commercially important species in N. America were identified as needing additional tests to determine the extent of any damage caused by infection.     

DNA addition or deletion is associated with a major karyotype polymorphism in the fungal phytopathogen Colletotrichum gloeosporioides

Summary A 1.2 Mb minichromosome resolved by pulsed-field electrophoresis was present in two independent race 3 isolates of Colletotrichum gloeosporioides causing Type B anthracnose specifically on Stylosanthes guianensis cv. Graham in Australia. This chromosome was absent in duplicate isolates representing races 1, 2 and 4 which infect other S. guianensis cultivars. A gene library was prepared specifically from the 1.2 Mb mini-chromosome and ten independent DNA clones unique to this chromosome were identified by differential hybridisation to whole chromosome probes. All of the ten selected probes hybridised only to the 1.2 Mb minichromosome unique to the race 3 isolates but not to any chromosome in isolates of the other races. These ten probes also hybridised only to restriction-digested DNA of race 3 and were thus both chromosome- and strain-specific for Type B C. gloeosporioides. Hybridisation analysis of NotI fragments of the 1.2 Mb minichromosome with these sequences indicated that they were not tightly clustered on the chromosome. These data demonstrate that the variation in the occurrence of the 1.2 Mb minichromosome did not arise by rearrangement of the genome of a progenitor strain but involved either large scale deletion or addition of DNA. The 1.2 Mb minichromosome did not contain a cloned high-copy-number repeat sequence present on all other mini- and maxichromosomes, suggesting addition from a genetically distinct strain. All ten chromosome-specific DNA probes hybridised to a 2.0 Mb chromosome in all races of C. gloeosporioides causing Type A anthracnose on Stylosanthes spp. including S. guianensis. Restriction fragment length polymorphism analysis demonstrated that only 15% of the hybridising restriction fragments of the Type A 2.0 Mb chromosome and the 1.2 Mb Type B race 3 minichromosome were identical. This indicated that it is unlikely that the 1.2 Mb minichromosome of the race 3 Type B pathogen was recently introgressed from-the Type A pathogen.     

Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides.

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.     

CgOpt1, a putative oligopeptide transporter from Colletotrichum gloeosporioides that is involved in responses to auxin and pathogeniCity

Background  

The fungus Colletotrichum gloeosporioides f. sp. aeschynomene produces high levels of indole-3-acetic acid (IAA) in axenic cultures and during plant infection. We generated a suppression subtractive hybridization library enriched for IAA-induced genes and identified a clone, which was highly expressed in IAA-containing medium.     

Aging of plasmodial heterokaryons in Didymium iridis

Summary Isogenic diploid plasmodia of different ages were produced by crossing clonal gamete populations of the myxomycete Didymium iridis. These plasmodia were then used to produce age heterokaryons to study the timing of death in comparison to the senescence of the two original homokaryons. A majority of the age heterokaryons died concurrently with the oldest nuclear component which indicates that the control of aging may be a dominant or active process in the true slime molds. Age heteroplasmons using transfilter contact between two plasmodia across 1 pore size Nucleopore membrane filters were also produced which indicates that senescence may not be a cytoplasmic infectious particle.     

Defence responses of chilli fruits to Colletotrichum capsici and Alternaria alternata

The induction of defence compounds and enzymes involved in the phenylpropanoid pathway were studied in the ripe and green chilli fruits inoculated with Colletotrichum capsici and Alternaria alternata. Total phenols and the activity of phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and catalase (CAT) increased in the inoculated ripe and green chilli fruits compared to the corresponding healthy fruits. Total phenols and the activities of the enzymes were at the maximum 2–3 d after inoculation and thereafter declined sharply in ripe chilli fruits, whereas slowly in green chilli fruits. In comparison with ripe chilli fruits, green chilli fruits were more resistant as they showed higher accumulation of total phenols and also higher activities of enzymes.     

Enhancement of disease caused by Colletotrichum truncatum in Sesbania exaltata by coinoculating with epiphytic bacteria

Colletotrichum truncatum isolate NRRL 13737 (ARS patent culture No. 18434) is being evaluated for development as a mycoherbicide against the problematic weed Sesbania exaltata. Studies were conducted to determine whether selected phylloplane microorganisms, used as coinoculants, could increase the severity of disease incited by C. truncatum in S. exaltata. Hemp Sesbania seedlings were grown in a variety of soils and environments, sprayed with conidia of C. truncatum, and the formation of appressoria was examined on leaves using epifluorescence microscopy. From hemp sesbania plants that supported high levels of appressoria formation, over 200 phyllosphere microorganisms were isolated. Fifteen of 73 microbial isolates assayed stimulated appressoria formation in vitro on cellophane membranes. Five of 8 superior isolates from the in vitro assay also enhanced disease symptoms induced by C. truncatum on S. exaltata compared to seedlings treated with conidia only. Populations of three selected superior isolates remained high on leaves during plant exposure to dew. The superior isolates initiated no apparent symptoms and rarely decreased seedling growth parameters in the absence of C. truncatum. This is the first instance of utilizing phylloplane microorganisms to increase the level of disease incited by a mycoherbicide agent. Microbial facilitators may provide a means of improving the weed control efficacy of mycoherbicides.     

Variation in encapsulation sensitivity of Cotesia sesamiae biotypes to Busseola fusca

Many braconid wasp species inject polydnaviruses to overcome their host's immune system. In the species Cotesia sesamiae, two biotypes exist that differ in their ability to develop in the host Busseola fusca. The biotype from coastal Kenya is infected with Wolbachia and is not able to develop in larvae of B. fusca, whereas the uninfected inland biotype of this wasp can develop in B. fusca. The genetic transmission of the developmental ability was studied through a series of genetic crosses and superparasitization experiments. The Wolbachia infection of the coastal type did not play a role in the encapsulation response of the host. Experiments show that the polydnaviruses of the wasps could not prevent the encapsulation of the coastal parasitoid eggs. Most likely, larval characteristics such as surface proteins played a more important role in the encapsulation response of the host even in the presence of a functional polydnavirus.     

Repair and mutation following UV damage in heterokaryons of Neurospora

Summary UV damage in a repair-deficient strain has been repaired when a repair-proficient strain has joined it in a heterokaryon. By substituting various repair-deficient mutants for the two components of this system, it has been shown that the rescue is accomplished by means of the excision repair system for pyrimidine dimers. Mutation has been monitored in the rescued nuclei and found to occur with a low frequency. When the rescuing component is replaced by a strain which lacks the excision repair system, there is a low frequency of rescue and the rescued nuclei have a high incidence of mutation. It is concluded that excision repair is relatively mutation-free, but in its absence another repair system is called upon which produces mutations.     

DNA synthesis and chromosome transfer inEscherichia coli K 12

Summary Two experiments were designed to test two alternative models of bacterial conjugation. According to the first model (Bouck andAdelberg, Nagata) the donor cell is ready for mating at the moment of completion of the chromosome replication.According to the second model (Jacob andBrenner) a special replication cycle is started during mating and the new chromosome enters the recipient cell at the same moment it was synthesized. In the first experiment synchronization ofHfr cells was used. It was shown that the efficiency of mating is constant during the whole life cycle. In the second experiment a pulse of radioactive phosphate was introduced into the mating medium. The zygotes were frozen in liquid nitrogen and the radioactive suicide of recombinants measured. Only those genetic markers, which were transmitted simultaneously with the radioactive pulse underwent radioactive suicide. Both experiments are in accord with the second model of conjugation.With 3 Figures in the Text