C. N. Mortensen

Identification and Characterisation of Xanthomonas campestris pv. campestris Strains from Tanzania by Pathogenicity Tests, Biolog, rep-PCR and Fatty Acid Methyl Ester Analysis

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a major disease constraint to cabbage production by smallholder farmers in Africa. Variability exists within the pathogen, and yet differentiation of Xcc strains from other closely-related xanthomonads attacking crucifers is often difficult. The Biolog system, fatty acid methyl ester analysis using microbial identification system (MIS), rep-PCR and pathogenicity tests were used to identify and characterise Xcc strains from Tanzania. Great diversity was observed among Xcc strains in their Biolog and rep-PCR profiles. Specific rep-PCR genomic fingerprints were linked to some geographical areas in the country. Most of the Xcc strains were clustered in two groups based on their fatty acid profiles and symptom expression in cabbage although some deviant strains were found. Each of the methods allowed a degree of identification from species, pathovar to the strain level. Biolog and MIS identified all Xcc strains at least to the genus level. Additionally, Biolog identified 47% of Xcc strains to the pathovar and 43% to strain level, whereas MIS identified 43% of the strains to pathovar level. In the absence of a database, the utility of rep-PCR for routine diagnosis of strains was limited, although the procedure was good for delineation of Xcc to the strain level. These findings indicate the existence of Xcc strains in Tanzania that are distinct from those included in Biolog and MIS databases. The limitations noticed warrant continued improvement of databases and inclusion of pathogenicity testing, using universally susceptible cultivars, as an integral part of strain identification.

Biological control of black rot (Xanthomonas campestris pv. campestris) of brassicas with an antagonistic strain of Bacillus subtilis in Zimbabwe

Biological control efficiency of an antagonistic, endophytic strain of Bacillus subtilis (strain BB) was evaluated against three strains of the black rot pathogen, Xanthomonas campestris pv. campestris (Xcc), in four Brassica crops (cabbage, cauliflower, rape and broccoli) grown during three consecutive growing seasons and on two soil types, in two different areas in Zimbabwe. Strain BB controlled the disease caused by strain Xcc B-147 in all Brassica crops during the dry and short rainy seasons. A similar effect was observed in cabbage using the strain Xcc 33908. Biological control was effective in broccoli, but not in cabbage and rape during the main rainy season in clay loam soil and limited biological control effect was still observed when these crops were grown in sandy loam soil. The endophytic colonisation of cabbage roots by strain BB was confirmed by immuno-blotting during the whole growing season. Biological control of black rot with strain BB is discussed in relation to its effect on Xcc strains, Brassica crops and to the effect of weather and soil conditions.

Bacterial wilt of tomato in Karnataka and its management by Pseudomonas fluorescens

Field surveys undertaken in major tomato growing districts of the Karnataka state, located in southern part of India, revealed a high incidence of bacterial wilt caused by Ralstonia solanacearum and it is one of the most destructive bacterial diseases of economically important crops. Across all the tomato cultivars under evaluation, the disease incidence in plants ranged from 9% to 39% whereas the incidence in seeds ranged from 4% to 18%. The effects of tomato seed treatments with Pseudomonas fluorescens in the control of bacterial wilt under greenhouse conditions revealed that the treatments protected plants against soil-borne infections of the bacterial wilt organism. Seed treatment with antagonistic P. fluorescens strain significantly improved the quality of seed germination and seedling vigour. The disease incidence was significantly reduced in plants raised from P. fluorescens treated seeds followed by challenge inoculation with R. solanacearum. Periodic field surveys for the incidence of bacterial wilt of tomato could be recommended to monitor the populations of the bacterial wilt pathogen. Workable measures are presented that could lead to the reduction of the prevalence of this serious disease in affected fields of the small farm-holders.

Prospects of molecular markers in Fusarium species diversity

Recent developments in genomics have opened up for newer opportunities to study the diversity and classification of fungi. The genus Fusarium contains many plant pathogens that attack diverse agricultural crops. Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium species still remains one of the most critical issues in fungal taxonomy, given that the number of species recognized in the genus has been constantly changing in the last century due to the different taxonomic systems. This review focuses of various molecular-based techniques employed to study the diversity of Fusarium species causing diseases in major food crops. An introduction of fusarial diseases and their mycotoxins and molecular-marker-based methods for detection introduce the concept of marker application. Various well-known molecular techniques such as random amplified polymorphic DNA, amplification fragment length polymorphism, etc. to more modern ones such as DNA microarrays, DNA barcoding, and pyrosequencing and their application form the core of the review. Target regions in the genome which can be potential candidates for generation of probes and their use in phylogeny of Fusarium spp. are also presented. The concluding part emphasizes the value of molecular markers for assessing genetic variability and reveals that molecular tools are indispensable for providing information not only of one Fusarium species but on whole fungal community. This will be of extreme value for diagnosticians and researchers concerned with fungal biology, ecology, and genetics.

Seed biopriming with novel strain of Trichoderma harzianum for the control of toxigenic Fusarium verticillioides and fumonisins in maize

Fusarium verticillioides is one of the most important fungal pathogens in maize causing both pre- and post-harvest losses and also capable of producing Fumonisins. In the present study attempts have been made for screening potential T. harzianum from native rhizosphere and to study its effect on Fusarium ear rot disease, fumonisin accumulation in different maize cultivars grown in India. Eight isolates of T. harzianum were isolated and T. harzianum isolate Th-8 exhibited better antifungal activity than carbendizim. Th-8 was formulated in different solid substrates like wheat bran, paddy husk, talcum powder and cornstarch. Maize seeds of kanchan (moderately resistant), pioneer (resistant) and sweet corn (susceptible) were selected for laboratory and field studies and these seeds were treated with a conidial suspension of T. harzianum at the rate of 1 × 108 spore/ml and formulation at the rate of 10 g/kg. Treated seeds were subjected to evaluate F. verticillioides incidence, seed germination, seedling vigour and field emergence, yield, thousand seed weight and fumonisin production. It was found that the pure culture of T. harzianum was more effective in reducing the F. verticillioides and fumonisin incidence followed by Talc formulation than the carbendizim treated and untreated control. Formulations of T. harzianum were effective at reducing the F. verticillioides and Fumonisin infection and also increasing the seed germination, vigour index, field emergence, yield, and thousand seed weight in comparison with the control.

Control of Fusarium verticillioides, cause of ear rot of maize, by Pseudomonas fluorescens

BACKGROUND Maize is one of the staple food crops grown in India. Fusarium verticillioides (Sacc.) Nirenberg is the most important fungal pathogen of maize, associated with diseases such as ear rot and kernel rot. Apart from the disease, it is capable of producing fumonisins, which have elicited considerable attention over the past decade owing to their association with animal disease syndromes. Hence, the present study was conducted to evaluate ecofriendly approaches by using a maize rhizosphere isolate of Pseudomonas fluorescens (Trev.) Mig. and its formulation to control ear rot disease and fumonisin accumulation, and also to study the capacity to promote growth and yield of maize. In vitro assays were conducted to test the efficacy of P. fluorescens as a seed treatment on seed germination, seedling vigour and also the incidence of F. verticillioides in different maize cultivars. The field trials included both seed treatment and foliar spray. For all the experiments, P. fluorescens was formulated using corn starch, wheat bran and talc powder. In each case there were three different treatments of P. fluorescens, a non-treated control and chemical control. RESULTS Pure culture and the formulations, in comparison with the control, increased plant growth and vigour as measured by seed germination, seedling vigour, plant height, 1000 seed weight and yield. P. fluorescens pure culture used as seed treatment and as spray treatment enhanced the growth parameters and reduced the incidence of F. verticillioides and the level of fumonisins to a maximum extent compared with the other treatments. CONCLUSION The study demonstrates the potential role of P. fluorescens and its formulations in ear rot disease management. The biocontrol potential of this isolate is more suited for fumonisin reduction in maize kernels intended for human and animal feed.

Chemical and Molecular Methods for Detection of Toxigenic Fungi and Their Mycotoxins from Major Food Crops

Mycotoxins are the secondary metabolites produced by certain molds on a wide range of agricultural commodities and are closely related to human and animal food chains. Mycotoxins are capable of causing disease in humans and other animals, and their detection is largely dependent on the sample matrix and the type of fungus causing their contamination. The strict regulations on trade of contaminated grains and seeds and other produce in industrial countries lead to economic burdens on farmers. In developing countries, the situation is aggravated where regulations may be nonexistent or not enforced and where consumption of home-grown cereals leads to a wide exposure to toxins. Important mycotoxins that occur quite often in food are deoxynivalenol/nivalenol, trichothecenes, zearalenone, ochratoxin A fumonisins, and aflatoxins. High concentrations of mycotoxins such as aflatoxins are consumed by humans in areas of the world with higher-than-average levels of liver cancer, childhood malnutrition, and disease. This chapter introduces rapid, robust, and user-friendly protocols currently applied in the identification of toxigenic fungi and important mycotoxins.

Assessment of strains of Pseudomonas syringae pv. tomato from Tanzania for resistance to copper and streptomycin

Abstract Fifty-six strains of Pseudomonas syringae pv. tomato (P.s. pv. tomato) were collected from tomato-producing areas in Tanzania and assessed for resistance to copper and antibiotics. The collection was done from three tomato-producing regions (Morogoro, Arusha and Iringa), representing three different ecological conditions in the country. After isolation and identification, the P. s. pv. tomato strains were grown on Kings medium B (KB) amended with 20% copper sulphate (w/v). The strains were also assessed for resistance to antibiotics. Results indicated that there was widespread resistance of the P. s. pv. tomato strains to copper sulphate. The highest level of resistance was recorded from the Arusha region (Northern Tanzania), 83.3% of the P. s. pv. tomato strains from that region showed resistance to copper sulphate. This was followed by Iringa region (Southern Tanzania), from where strains of the pathogen were moderately resistant to copper sulphate, such that 54.0% of them were able to grow on the KB medium amended with 20% (w/v) of the copper compound. Out of seven strains of P. s. pv. tomato from Morogoro region (Central Tanzania) included in the study, five (71.5%) were resistant to copper sulphate. The only strain of P. s. pv. tomato from the Dodoma region (Central Tanzania, but with a different ecological condition from the Morogoro region) included in the study was unable to grow on the medium containing 20% copper sulphate. None of the P. s. pv. tomato strains in the four regions included in the study were resistant to streptomycin sulphate. These results suggest that in the Arusha and Iringa regions of Tanzania, there might be possibilities of excessive use of copper compounds in tomato production, such that strains of P. s. pv. tomato strains in the areas have become resistant to the compounds.

Xanthomonads and other yellow-pigmented Xanthomonas-like bacteria associated with tomato seeds in Tanzania

Tomato ( Solanum lycopersicum L.) seeds habour unique bacterial community that can be pathogenic or beneficial to their host. Xanthomonas causing bacterial leaf spot (BLSX) on tomato and other yellow-pigmented xanthomonads-like bacteria (XLB) that closely resemble BLSX were obtained from tomato seeds collected from Northern, Central and Southern highland regions of Tanzania. A total of 73 strains were isolated from 52 seed samples of 15 tomato cultivars. Results obtained with Biolog and sequence analysis of the 16S rRNA gene showed that samples originating from Central Tanzania harbored the most diverse populations of XLB and BLSX as compared to Northern and Southern Tanzania. The predominant bacterial genera in tomato seeds were Stenotrophomonas , Sphingomonas , Chryseobacterium , Xanthomonas , Pantoea and Flavobacterium . All strains identified by Biolog as Xanthomonas with exception of Xanthomonas campestris pv. malvacearum , were pathogenic on tomato and pepper plants. Strains identified by Biolog as Sphingomonas sanguinis and Sphingomonas terrae also incited black rot symptoms on pepper leaves. However, bacterial strains belonging to the genus Stenotrophomonas , Chryseobacterium , Pantoea and Flavobacterium were not pathogenic on tomato and pepper. Phylogenetic analysis showed that strains of the genus Xanthomonas are more closely related to Stenotrophomonas and Pantoea compared to the other bacterial genera found in tomato seeds. Key words: Xanthomonas , yellow-pigmented bacteria, seed, tomato, phylogeny.

Molecular Characterization of Pseudomonas syringae pv. tomato Isolates from Tanzania

Bacterial speck caused byPseudomonas syringae pv.tomato is an emerging disease of tomato in Tanzania. Following reports of outbreaks of the disease in many locations in Tanzania, 56 isolates ofP. syringae pv.tomato were collected from four tomato- producing areas and characterized using pathogenicity assays on tomato, carbon source utilization by the Biolog Microplate system, polymerase chain reaction and restriction fragment length polymorphism (RFLP) analysis. All theP. syringae pv.tomato isolates produced bacterial speck symptoms on susceptible tomato (cv. ‘Tanya’) seedlings. Metabolic fingerprinting profiles revealed diversity among the isolates, forming several clusters. Some geographic differentiation was observed in principal component analysis, with isolates from Arusha region being more diverse than those from Iringa and Morogoro regions. The Biolog system was efficient in the identification of the isolates to the species level, as 53 of the 56 (94.6%) isolates ofP. syringae pv.tomato were identified asPseudomonas syringae. However, only 23 isolates out of the 56 (41.1%) were identified asPseudomonas syringae pv.tomato. The results of this work indicate the existence ofP. syringae pv.tomato isolates in Tanzania that differ significantly from those used to create the Biolog database. RFLP analysis showed that the isolates were highly conserved in theirhrpZ gene. The low level of genomic diversity within the pathogen in Tanzania shows that there is a possibility to use resistant tomato varieties as part of an effective integrated bacterial speck management plan.