Vegetative Compatibility Grouping of Colletotrichum kahawae in Kenya

Vegetative compatibility using nitrate nonutilizing ( nit ) mutants was analysed between 44 isolates of Colletotrichum kahawae from Kenya, one each from Ethiopia and Malawi, one of Colletotrichum gloeosporioides and one of Colletotrichum acutatum . Another isolate of C. kahawae did not generate mutants and thus could not be utilized. The results showed that all the C. kahawae isolates, except a white sector mutant (VCG2), belonged to one vegetative compatibility group (VCG4). The other species belonged to their own unique groups (VCGs 1 and 3). Implications of the results and future research needs on the subject are discussed.     

Variability of Colletotrichum kahawae in Relation to Other Colletotrichum Species from Tropical Perennial Crops and the Development of Diagnostic Techniques

Twenty‐six isolates of Colletotrichum kahawae, the causal agent of coffee berry disease, from coffee in Africa, and 25 isolates, mostly of Colletotrichum gloeosporioides, from coffee and other tropical perennial crops, were examined for the ability to metabolize citrate and tartrate and their molecular genetic variability was assessed using restriction fragment length polymorphisms (RFLP) and variable number tandem repeats (VNTR). Twenty‐four isolates of C. kahawae were also assessed using amplified fragment length polymorphisms (AFLP). Vegetative compatibility within a collection of nine isolates, including two of C. gloeosporioides was also assessed. All isolates of C. kahawae from across Africa failed to metabolize citrate or tartrate, but all other isolates metabolized one or both. Colletotrichum kahawae isolates also showed minimal variability using the molecular techniques with two isolates from Cameroon showing slightly different banding patterns in RFLP analysis. All other isolates had variable VNTR and RFLP banding patterns. AFLP analysis failed to detect variability within 12 isolates from Kenya, but did detect differences between isolates from other countries. Five isolates from Kenya were vegetatively compatible but differed from two from Cameroon and from two C. gloeosporioides isolates. Results demonstrate some geographic variability within C. kahawae isolates, although this is small, probably due to the relatively young age of C. kahawae populations. The biochemical and molecular techniques used showed clear differences from other Colletotrichum isolates, and can be used to distinguish the species. Lack of citrate and tartrate metabolism provides a readily applicable diagnostic method.     

Infection biology and defence responses in sorghum against Colletotrichum sublineolum

Aims:  To investigate the infection biology of Colletotrichum sublineolum (isolate CP2126) and defence responses in leaves of resistant (SC146), intermediately resistant (SC326) and susceptible (BTx623) sorghum genotypes.
Methods and Results:  Infection biology and defence responses were studied quantitatively by light microscopy, H₂O₂ accumulation by DAB staining and HRGP accumulation by immunological methods. Inhibition of conidial germination and appressorium formation may represent prepenetration defence responses on the leaf surface. Inducible defence responses in the resistant genotypes included decreases in formation of appressoria as well as accumulation of H₂O₂, HRGPs and phytoalexins. Concomitant with these inducible responses, fungal growth was stopped during or just after penetration in genotypes SC146 and SC326. High levels of H₂O₂ accumulating at late infection stages (5 days after inoculation) in the susceptible genotype BTx623 correlated with necrosis and tissue degeneration.
Conclusions:  The early accumulation of H₂O₂ and HRGPs indicates roles in defence whereas the late accumulation in genotype BTx623 correlated with successful pathogenesis.
Significance and Impact of the Study:  The fact that there is a significant correlation between induced accumulation of H₂O₂, papilla formation and cell wall cross-linking, as evidenced by HRGP accumulation, and cessation of pathogen growth in resistant genotypes may help exploit host resistance in sorghum.     

Accumulation of trans-piceid in sorghum seedlings infected with Colletotrichum sublineolum

Sorghum SbSTS1, a pathogen inducible gene, was previously demonstrated to encode an enzyme with stilbene synthase activity. In this study, we attempt to identify the stilbene derivatives that accumulate in infected sorghum seedlings after inoculation with the anthracnose pathogen Colletotrichum sublineolum. Scanning for precursor ions that produced the common stilbene aglycones as diagnostic ions was performed in a triple quadrupole mass spectrometer. It was found that infected sorghum seedlings accumulated trans-piceid as the major stilbene metabolite together with an unknown resveratrol derivative. Time-course accumulation of trans-piceid was examined in two sorghum cultivars, DK18 and DK77, which are resistant and susceptible to C. sublineolum, respectively. In both cultivars, trans-piceid was not detected until 48h after inoculation, consistent with the late induction of SbSTS1 reported previously in infected sorghum plants. The levels of trans-piceid detected in DK77 seedlings were approximately three times the levels detected in DK18 seedlings at 120h after inoculation. In vitro assays demonstrated that trans-piceid did not exhibit significant toxicity on conidial germination and mycelial growth of C. sublineolum. Hence trans-piceid alone may not represent an important defense component against the anthracnose pathogen in sorghum seedlings.     

Vegetative Compatibility Among Isolates of Colletotrichum gloeosporioides from Yam (Dioscorea spp.) in Nigeria

Isolates of Colletotrichum gloeosporioides obtained from yam‐based cropping systems in Nigeria, previously characterized on the basis of morphology, virulence and rDNA internal transcribed spacer (ITS) sequence variation were further compared for vegetative compatibility (VC). Chlorate‐resistant nitrate non‐utilizing (nit) mutants were generated from the isolates and used in complementation (heterokaryon) tests. Tests of VC between complementary mutants from different isolates indicated the presence of several genotypes within a single field, suggesting limited clonal spread. In some cases, isolates obtained from the same lesion were observed to belong to different vegetative compatibility groups (VCGs). No compatibility was observed between isolates of the highly virulent slow‐growing grey (SGG), the moderately virulent fast‐growing salmon (FGS) and the avirulent/weakly virulent fast‐growing grey (FGG) strains. Forty‐one C. gloeosporioides isolates belonged to 28 VCGs, giving a genotype diversity estimate of 0.68. This diversity confirmed the high variability of the pathogen population as revealed by previous characterization studies, however, a correlation between VCGs and isolate groupings based on morphology and virulence was not found. The finding that an isolate from weed was compatible with yam isolates indicated that transfer of important traits, such as virulence, may take place between isolates from yam and non‐yam hosts. The VCG diversity revealed by this study suggests that in addition to asexual reproduction, sexual reproduction may play an important role in the epidemiology of anthracnose on yam.     

Genetic diversity of Colletotrichum gloeosporioides species complex associated with Citrus wither‐tip of twigs in Tunisia using microsatellite markers

Anthracnose Citrus disease has been associated with several symptoms worldwide and it is recently compromising Citrus production in the Mediterranean area. Four species complexes are mainly involved: Colletotrichum boninense, C. acutatum, C. gloeosporioides and C. truncatum. In this study, we investigated the genetic diversity of Colletotrichum spp. in Tunisia associated with wither‐tip of twigs on Citrus. Specific primers ITS4‐CgInt allowed the identification of C. gloeosporioides species complex in all the 54 isolates, sampled from three regions and four Citrus species. Overall, our genotypic analysis using 10 SSR markers showed a moderate diversity level in Tunisian C. gloeosporioides population and highlighted that C. gloeosporioides reproduce mainly clonally. In addition, heterothallic isolates were present in our population, suggesting that the pathogen population may undergo parasexual recombinations. The highest genetic diversity in C. gloeosporioides was recorded in Nabeul and on orange, which likely constitutes the area and the host of origin for the Citrus anthracnose disease in Tunisia. In addition, no population subdivision was detected at the geographic, host species or cultivars’ origin levels. However, our study identified two genetic subpopulations and indicated a rapid C. gloeosporioides population change at temporal scale that should be further examined over several consecutive growing seasons in order to understand its population dynamics.     

Pathotypes of Colletotrichum sublineolum in Response to Sorghum Populations with Different Levels of Genetic Diversity in Sete Lagoas‐MG

Sorghum anthracnose is one of the most important and destructive diseases of sorghum. Genetic resistance has been the most efficient strategy to control the disease, but the high variability of the pathogen population in Brazil has resulted in only modest efficacy. Accordingly, we investigated the variability of Colletotrichum sublineolum in response to sorghum populations with three levels of genetic diversity: pure stand, three‐way hybrids and physical mixtures of three‐way hybrids. Six plots of each treatment were planted in different areas and at different dates. A total of 480 isolates, that is 40 single‐conidium isolates per plot, were collected from the field experiment to characterize the variability of the pathogen in each host population. Isolates were inoculated in a greenhouse on a differential line set composed of eight sorghum inbred lines. Our results reveal that the pathogen populations derived from three‐way combinations had higher pathotype diversity than did those derived from pure stand host populations. More complexly, virulent phenotypes were also developed in genetically diverse stands compared to pure stand host populations. The diversification of the host population limits pathogen adaptation, thus resulting in a significantly higher number of pathotypes. The results of this study will improve the management of sorghum anthracnose in the field by helping sorghum breeders maintain disease resistance.     

Genetic Recombination in Micromonospora

Biochemical mutants were obtained from Micromonospora chalcea, M. purpurea, and M. echinospora by using ultraviolet radiation or nitrosoguanidine. Crosses carried out between complementary nutritional mutants of the same species showed positive genetic interaction. Data are reported which indicate that the interaction between the crossed strains is due to genetic recombination. No evidence for interspecific genetic recombination was found.     

Genetic Recombination in Mycobacteria

Evidence for genetic recombination between Mycobacterium smegmatis strain Rabinowitchi (Rab) and strain Jucho or PM5 is presented. Backcrosses of recombinants by either parental strain indicated four different types of mating behavior, suggesting that the mycobacterial compatibilities are controlled by at least two different factors. No sex factor that transfers at a high frequency or that is sensitive to acridine dyes was detected. Analysis of segregation of unselected markers revealed that strain Jucho, or PM5, contributes the majority of alleles in almost all recombinants obtained from different selective media. Efforts to construct linkage maps for the markers employed failed because of ordering ambiguities. Mating medium containing streptomycin prevented genetic recombination when strain Rab was resistant to the antibiotic and Jucho, or PM5, was sensitive, but it did not prevent recombination when Rab was sensitive to streptomycin and Jucho, or PM5, was resistant. Very low frequency of recombinant formation was observed when Jucho, or PM5, had been treated with streptomycin, whereas recombinants were formed at fairly high frequencies when Rab had been treated with the antibiotic, suggesting that the roles of parental strains in zygote formation were not identical. The results suggest a polar transfer of genetic material from Rab to Jucho, or PM5, although an alternative possibility of cell fusion followed by exclusion could not be excluded.     

Modeling Interference in Genetic Recombination

In analyzing genetic linkage data it is common to assume that the locations of crossovers along a chromosome follow a Poisson process, whereas it has long been known that this assumption does not fit the data. In many organisms it appears that the presence of a crossover inhibits the formation of another nearby, a phenomenon known as ``interference.' We discuss several point process models for recombination that incorporate position interference but assume no chromatid interference. Using stochastic simulation, we are able to fit the models to a multilocus Drosophila dataset by the method of maximum likelihood. We find that some biologically inspired point process models incorporating one or two additional parameters provide a dramatically better fit to the data than the usual ``no-interference' Poisson model.     

Sexual Isolation and Genetic Diversification Among Some Strains of Crypthecodinium cohnii-like Dinoflagellates Evidence of Speciation

SYNOPSIS. Nine new isolates of Crypthecodinium-like dinoflagellates from diverse geographic locations, together with 3 established strains from Woods Hole and Puerto Rico, were analyzed for sexual compatibility by means of a complementation test using motility mutants. The results indicate that 5 of the 12 are mutually compatible and thereby represent one species. Five others are clearly reproductively isolated from this group and from each other and therefore may belong to separate species. The position of 2 other isolates remains uncertain. Only one, MC-5, is markedly distinct morphologically from all the others. It is also sympatric with one of the others, strain G, and its separation from Crypthecodinium cohnii seems therefore more fully justified than that of the other sexually isolated strains. G and MC-5 would be considered "good" species by an evolutionary biologist. The others are from widely separate geographic origins and. though still poorly characterized, all superficially resemble C. cohnii . Comparison of many characters (DNA profile, radiation response, drug sensitivity, macroalgal association, etc.) of the incompatible strains show significant disparities which are discussed in considering speciation.     

Assessment of sorghum genetic resources of Ethiopia for anthracnose (Colletotrichum sublineolum Henn.) resistance and agronomic traits

Sorghum anthracnose caused by Colletotrichum sublineolum Henn. is one of the key diseases limiting sorghum production and productivity. Development of anthracnose‐resistant sorghum genotypes possessing yield‐promoting agronomic traits is an important breeding goal in sorghum improvement programs. The objective of this study was to determine the responses of diverse sorghum genetic resources for anthracnose resistance and agronomic traits to identify desirable lines for breeding. A total of 366 sorghum collections and three standard checks were field evaluated during the 2016 and 2017 cropping seasons. Lines were artificially inoculated with a virulent pure isolate of the pathogen. Anthracnose disease severity was assessed to calculate the area under disease progress curve (AUDPC). Agronomic traits such as panicle length (PL), panicle width (PW), head weight (HW) and thousand grain weight (TGW) were measured. Lines showed highly significant differences (p < .001) for anthracnose severity, AUDPC and agronomic traits. Among the collections 32 lines developed levels of disease severity between 15% and 30% in both seasons. The following sorghum landraces were selected: 71708, 210903, 74222, 73955, 74685, 74670, 74656, 74183, 234112, 69412, 226057, 214852, 71420, 71484, 200126, 71557, 75120, 71547, 220014, 228179, 16212, 16173, 16133, 69088, 238388, 16168 and 71570. These landraces had a relatively low anthracnose severity possessing farmer‐preferred agronomic traits. The selected genotypes are useful genetic resources to develop anthracnose‐resistant sorghum cultivars.     

Genetic diversity within and among vegetative compatibility groups of Stereum sanguinolentum determined by arbitrary primed PCR

Genetic variation within and among vegetative compatibility groups (VCGs) of Stereum sanguinolentum isolates was investigated with various geographical distances. DNA fingerprints were made using the M13 core sequence as a primer. A total of 113 isolates from 12 plots in Sweden, Finland and Lithuania were studied. Each VCG produced a discrete group of banding patterns. Among 20 isolates from the largest VCG, G1, the incidence of identical banding patterns was 24% within a sample plot, 8% among sample plots within a country, and 0% among countries. In the other 15 VCGs that comprised two and more isolates, corresponding percentages were 42%, 30% and 0%. Average band-sharing indices (ABSIs) within VCGs decreased with increasing geographical distance: for isolates from VCG G1, ABSI was 91.3% within sample plots, 85.4% among sample plots within a country, and 79.2% among countries. Corresponding figures for the other 15 VCGs were 97.0%, 92.7% and 80.4%, respectively. Among VCGs similarities were significantly lower ( P < 0.001) and the trend of decreasing similarity with larger geographical distances was less pronounced: ABSI within sample plots was 73.7%, among sample plots within countries 73.8%, and among countries 71.1%. However, the similarity of populations as compared within and between countries differed significantly ( P < 0.001), suggesting geographical differentiation between S. sanguinolentum populations separated by 400 km and the Baltic sea. Calculations of G _ST clearly indicated geographical population subdivision within the large VCG but not among the total sample. In summary, the results show population structure of inbred lines within VCGs containing closely related strains in local populations and more distant relationships among populations.     

Genetic isolation among sympatric vegetative compatibility groups of the aflatoxin‐producing fungus Aspergillus flavus

Aspergillus flavus, a fungal pathogen of animals and both wild and economically important plants, is most recognized for producing aflatoxin, a cancer‐causing secondary metabolite that contaminates food and animal feed globally. Aspergillus flavus has two self/nonself recognition systems, a sexual compatibility system and a vegetative incompatibility system, and both play a role in directing gene flow in populations. Aspergillus flavus reproduces clonally in wild and agricultural settings, but whether a cryptic sexual stage exists in nature is currently unknown. We investigated the distribution of genetic variation in 243 samples collected over 4 years from three common vegetative compatibility groups (VCGs) in Arizona and Texas from cotton using 24 microsatellite loci and the mating type locus (MAT) to assess population structure and potential gene flow among A. flavus VCGs in sympatric populations. All isolates within a VCG had the same mating type with OD02 having MAT1‐2 and both CG136 and MR17 having MAT1‐1. Our results support the hypothesis that these three A. flavus VCGs are genetically isolated. We found high levels of genetic differentiation and no evidence of gene flow between VCGs, including VCGs of opposite mating‐type. Our results suggest that these VCGs diverged before domestication of agricultural hosts (>10 000 yr bp ).     

Isolation and characterization of cAMP unresponsive (frigid) aggregation-deficient mutants of Dictyostelium discoideum

To find mutants of Dictyostelium discoideum that are unable to respond to exogenous cAMP signals (frigid mutants), amoebae of 218 independent aggregation-deficient mutants were treated in suspension with artificial pulses of cAMP and screened for the capacity to form EDTA-resistant cohesion sites. Eleven frigid mutants were identified and further characterized. Using parasexual genetic techniques, these strains were assigned to five complementation groups (fgdA-E) and the fgd loci were mapped in three linkage groups: fgdA and D in group II, fgdC in group III, and fgdB and E in group VII. Biochemical and physiological experiments with these strains indicated that fgd mutants are of two general types. When starved, strains in groups fgdB, D, and E failed to produce detectable levels of membrane-associated cAMP phosphodiesterase, surface cAMP receptors, or extracellular phosphodiesterase inhibitor, and the cells continued to respond chemotactically to folate. Thus, these strains are probably arrested in the vegetative phase or very early in development. In contrast, strains in groups fgdA and C produced low levels of cAMP receptors and secreted phosphodiesterase inhibitor. Moreover, after starvation, some of these mutants elicited a weak chemotactic response to cAMP. Therefore, unlike the former group of mutants, these strains appear to initiate development when starved, but the process is blocked at an early stage.     

Races and virulence of Fusarium oxysporum f.sp. cubense on local banana cultivars in Kenya

Virulence of 31 Kenyan isolates of Fusarium oxysporum obtained from bananas showing symptoms of Panama disease was tested against the differential banana cvs Bluggoe, Gros Michel, Dwarf Cavendish, and two other local cvs Muraru and Wang'ae. Seventeen isolates were assigned to either race 1 or race 2 of F. oxysporum f.sp. cubense (FOC). Race 4 was not apparent in this sample of 31 isolates from Kenya as none were pathogenic to cv. Cavendish, and no wilted Cavendish have been observed in field surveys in Kenya. Races could not be assigned to 12 isolates as they were virulent on more than one differential cultivar, and two were apparently not pathogenic. All isolates assigned to races 1 and 2 belonged to the VCG bridging complex 0124/5/8/20, but some other isolates belonging to this VCG complex could not be assigned to race. All five isolates assigned to VCG 01212 could not be assigned to known races. Considerable variability thus exists within FOC isolates within this region. Local cultivars of banana showed differential resistance to the pathogen. The interaction of cultivars and isolates on the level of disease was significant. Overall, cv. Wang'ae was the most susceptible to most of the isolates tested, regardless of their race, and could therefore be used as a reference cultivar in pathogenicity tests of isolates of FOC in the East African region. Of the cultivars tested that are widely grown on smallholder farms in Kenya, Muraru was the least susceptible.