Development of a semiselective medium for isolating Xanthomonas campestris pv. musacearum from insect vectors, infected plant material and soil

A semiselective medium was developed for isolating Xanthomonas campestris pv. musacearum ( Xcm ) from infected banana plants, soil and insect vectors. The new medium was named cellobiose-cephalexin agar (CCA) and it contained (L⁻¹): 1 g yeast extract, 1 g glucose, 1 g peptone, 1 g NH₄Cl, 1 g MgSO₄·7H₂O, 3 g K₂HPO₄, 1 g beef extract, 10 g cellobiose, 14 g agar, 40 mg cephalexin, 10 mg 5-fluorouracil and 120 mg cycloheximide. The medium was evaluated for selectivity using 21 bacterial isolates and for plating efficiency using Xcm . The bacterial isolates included a soilborne Xanthomonas species and three pathogenic Xanthomonas strains that infect cassava, cabbage and beans. Although the plating efficiency of Xcm on CCA was lower (59%) than on non-selective yeast extract peptone glucose agar (YPGA), its selectivity was significantly higher, averaging 60 and 82%, when isolating from banana fruits and soil, respectively. CCA was also superior when isolating Xcm from insect vectors, with selectivity of 48–75%, compared with 8–17% on YPGA. Xanthomonas campestris pv. phaseoli did not grow on CCA, while X. campestris pv. campestris and X. axonopodis pv. manihotis grew, but their colonies were smaller than those of Xcm . Twenty-nine out of 33 suspected Xcm strains isolated from plants, soil and insects using CCA were pathogenic when inoculated onto banana plants, indicating that CCA can be a reliable tool in isolating Xcm populations. The medium should prove useful in studies on ecology, epidemiology and management of the banana bacterial wilt pathogen that is currently ravaging bananas in East and Central Africa.     

Inheritance of resistance to Fusarium oxysporum f. sp. cubense race 1 in bananas

Fusarium wilt of bananas (also known as Panama disease), caused by the soil-borne fungus Fusarium oxysporum f. sp cubense (Foc), is a serious problem to banana production worldwide. Genetic resistance offers the most promising means to the control of Fusarium wilt of bananas. In this study, the inheritance of resistance in Musa to Foc race 1 was investigated in three F₂ populations derived from a cross between ‘Sukali Ndizi’ and ‘TMB2X8075-7’. A total of 163 F₂ progenies were evaluated for their response to Fusarium wilt in a screen house experiment. One hundred and fifteen progenies were susceptible and 48 were resistant. Mendelian segregation analysis for susceptible versus resistant progenies fits the segregation ratio of 3:1 (χ² = 1.72, P = 0.81), suggesting that resistance to Fusarium wilt in Musa is conditioned by a single recessive gene. We propose panama disease 1 to be the name of the recessive gene conditioning resistance to Fusarium wilt in the diploid banana ‘TMB2X8075-7’.     

Contribution of synthetic tetraploids (AAAA) and diploids (AA) to black Sigatoka resistance and bunch weight to their triploid progenies

The generation of banana triploids from tetraploid-diploid crosses requires knowledge on the influence of the parents on black Sigatoka resistance and agronomic traits to the triploid progenies. The objective of this investigation was to determine the influence of tetraploid and diploid parents on black Sigatoka resistance and agronomic traits in the triploid progenies generated from tetraploid-diploid crosses. The mating scheme was designed as a 4 × 5 North Carolina II mating design. Due to problems in seed set and germination, progenies from 2 male parents with 4 female parents were evaluated at two sites in Uganda. The results showed that the male-parent triploid progeny heritability estimate for the number of leaves at harvest was greater than the female parent estimate. The diploid parents had higher correlation coefficients for the total leaves at harvest with the triploid progenies than tetraploid parents with triploid progenies. Disease development over time took more days in diploid parents than in the tetraploid parents with the triploid progenies as intermediates. These results suggested that diploids transferred black Sigatoka resistance to the triploid progenies as measured by the number of standing leaves and disease development overtime. There was a positive correlation (P < 0.05) between tetraploid female parents and triploid progenies for plant height and bunch weight. The triploid progeny-tetraploid female parent heritability estimates for plant height (0.92) and bunch weight (0.72) were highly significant. These results indicated that the female synthetic tetraploids influenced plant height and bunch weight in the triploid progenies. Therefore, it is important to select the tetraploids with heavy bunches to effectively improve yield in triploid progenies generated by tetraploid-diploid crosses. The tetraploid-diploid progenies had a significant (P < 0.05) family-by-site interaction for bunch weight indicating that new banana genotypes need to be tested across different environments to select stable genotypes to promote to end-users.     

Development of a semi-selective medium for isolation of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv<Emphasis Type="Italic">. musacearum</Emphasis> from banana plants

Banana Xanthomonas wilt, caused by Xanthomonas campestris pv. musacearum, is a new threat to banana cultivation in eastern Africa. The causal bacterium grows slowly in culture and is easily overgrown by contaminants. A selective culture medium for isolation of X. c. pv. musacearum will facilitate disease study. A medium that suppressed saprophytic growth and possessed diagnostic characters for the pathogen was developed. Various carbon sources were tested with two isolates of X. c. pv. musacearum, and sucrose was selected as main carbon source. The susceptibility of X. c. pv. musacearum and other bacterial strains was tested with 29 different antibiotics. Cephalexin and cycloheximide had no effect on X. c. pv. musacearum but cephalexin inhibited most of the saprophytes and cycloheximide inhibited the fungal contaminants. Based on these studies, we have developed a semi-selective medium YTSA-CC containing yeast extract (1%), tryptone (1%), sucrose (1%), agar (1.5%), cephalexin (50 mg l⁻¹) and cycloheximide (150 mg l⁻¹), pH 7.0. The pathogen X. c. pv. musacearum was easily identified as yellowish, mucoid and circular colonies on YTSA-CC medium. This simple semi-selective medium was effective for isolation of X. c. pv. musacearum from infected banana tissues and soil, and it should be a valuable tool in ecological and epidemiological studies.     

Agricultural management affects communities of culturable root-endophytic fungi in temperate grasslands

Three grassland sites of similar physical characteristics but differing management histories were chosen to test the hypothesis that agricultural disturbance has a deleterious effect on the diversity of culturable root-endophytic fungi and favours potentially pathogenic species. Species abundance data were collected for fungi isolated from surface sterilised root samples. Brillouin index of diversity, Twinspan and detrended correspondence analysis were applied to the community data. Quantitative ordination separated the samples by site showing that the communities differed in fields of contrasting management and this was supported by data from a microcosm experiment. Species presence and absence appeared to be affected seasonally; site differences were manifested in relative abundance. Diversity did not appear to vary by site, but a methodological explanation for this is proposed. Sterile dark septate endophytes were shown to be among the most abundant groups at all sites.     

Improved PCR for identification of members of the genus Xanthomonas

A PCR-based system was developed to reliably and robustly identify group I and II members of the genus Xanthomonas. Primer sets developed from three gene targets namely fyuA, ITS and gumD were evaluated in the study. Primer sets were evaluated using DNA extracted from 45 Xanthomonas strains representing 25 species broadly covering the genus. Fifteen non-Xanthomonas strains of plant-associated bacteria including phylogenetically closely related species Stenotrophomonas maltophilia and Xylella fastidiosa were also tested. The primers targeting fyuA amplified DNA from all xanthomonads except X. theicola, while the ITS primers amplified a DNA fragment of 254 bp in all 45 Xanthomonas strains; whereas no amplification was observed for non-xanthomonads. The gumD primers allowed efficient amplification of DNA in 38 out of 39 isolates from Group II, whereas no or very weak amplification occurred with DNA from Group I members. Internal controls of primers targeting bacterial 16S rDNA or plant 26S mitochondrial rDNA were successfully applied in multiplex PCRs for testing bacterial cultures or plant tissue, respectively. The findings give us a PCR based approach that can reliably and effectively differentiate xanthomonads from non-xanthomonads as well as separating the strains belonging to the two described groups of the genus Xanthomonas. The study thus offers valuable tools for disease surveillance and management. It can effectively be applied in rapid assessment of new disease occurrences, for which no specific detection tools could be in place.     

Characterization of the Xanthomonas sp. causing wilt of enset and banana and its proposed reclassification as a strain of X. vasicola

Comparative analyses were undertaken to characterize Xanthomonas campestris pv. musacearum, the causal agent of a wilt of enset and banana, and to assess its relatedness to other xanthomonads by fatty acid methyl esters, genomic fingerprinting using rep-PCR and partial nucleotide sequencing of the gyrase B gene. The results from all three analyses indicated that strains of X. campestris pv. musacearum are homogeneous and very similar to X. vasicola strains isolated from sugarcane and maize from Africa. Pathogenicity studies indicated that strains of X. vasicola pv. holcicola and X. vasicola from sugarcane induced no symptoms on banana, whereas X. campestris pv . musacearum produced severe disease. These data will support a future proposed reclassification of X. campestris pv. musacearum as X. vasicola pv . musacearum when more data are available.     

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The present study developed a pathovar‐specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265‐bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant‐associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm‐specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.