Fen Beed

Integrating Fusarium oxysporum f. sp. strigae into cereal cropping systems in Africa

BACKGROUND Striga hermonthica (Del.) Benth. (witchweed) poses the greatest biological constraint to food production in sub-Saharan Africa (SSA). Control options for Striga are currently largely ineffective or unavailable to farmers, and other management possibilities are urgently needed. Biological control obviates some of the problems of several of the other techniques and provides a management option that is durable and environmentally responsive. The efficacy of S. hermonthica control using different formulations of three isolates of Fusarium oxysporum Schlecht. emend. Synder & Hans f. sp. strigae was tested on Striga-resistant and Striga-susceptible varieties of sorghum and maize under African field conditions for the first time. RESULTS Isolates PSM197 and Foxy 2 were effective in witchweed repression, especially when applied as pesta granules. Isolate M12-4A was less effective under the field conditions investigated. Application of the fungi was generally more beneficial in maize than in sorghum for the varieties tested. Application of the biocontrol agent caused significant decreases in the number of flowering Striga plants, and hence deposition of seeds with impact of enhancing future crop yield. CONCLUSIONS Synergistic effects between the Striga-resistant maize line and Fusarium oxysporum f. sp strigae led to over 90% reduction in Striga emergence. These results will further encourage the distribution of the isolates tested or selection of country-specific relatives as viable and environmentally safe biocontrol agents to be used against Striga. Pesta was the most effective formulation, while seed coating may be more cost effective.

Fine-tuning banana Xanthomonas wilt control options over the past decade in East and Central Africa

Xanthomonas wilt, caused by Xanthomonas campestris pv. musacearum has, since 2001, become the most important and widespread disease of Musa in East and Central Africa. Over the past decade, new research findings and especially feedback from small-scale farmers have helped in fine-tuning Xanthomonas wilt control options. During the initial years of the Xanthomonas wilt epidemic in East Africa, the complete uprooting of diseased mats and the burning or burying of plant debris was advocated as part of a control package which included the use of clean garden tools and early removal of male buds to prevent insect vector transmission. Uprooting a complete mat (i.e. the mother plant and a varying number of lateral shoots) is understandably time-consuming and labour intensive and becomes very cumbersome when a large number of diseased mats have to be removed. Recent research findings suggest that Xcm bacteria do not colonize all lateral shoots (i.e. incomplete systemicity occurs) and even when present that this does not necessarily lead to symptom expression and disease. This led to a new control method whereby only the visibly diseased plants within a mat are cut at soil level. The underlying idea is that the continued removal of only the diseased plants in a field will reduce the inoculum level and will bring down disease incidence to an acceptable level. This method is less labour intensive and takes a short time compared to the removal of a complete mat. However, single diseased stem removal needs to go hand in hand with prevention of new infections that can occur through the use of contaminated garden tools or through insect vector transmission. Novel transgenic approaches are also discussed. This paper presents an overview of past and ongoing research towards the development of a more practical and less demanding control strategy for Xanthomonas wilt.

Managing the biological environment to promote and sustain crop productivity and quality.

Global crop production needs to double by 2050 in order to meet demands from rising populations, diet shifts and biofuels. Production must increase through more efficient use of currently available arable land to prevent encroachment on land that otherwise provides vital services to the earth and its people (i.e. through increased biodiversity and reduced carbon emissions). Significant improvements can be realised through enhanced management of critical diseases of crops that are pivotal to food security and income generation. To achieve this, the dynamic and complex interactions between crops and beneficial or antagonistic organisms that characterise the biological environment, must be understood. For Sub-Saharan Africa (SSA) a crop healthcare system is required that encompasses national responsibility and regional cooperation, and which harnesses global excellence in terms of the knowledge and methods that are available for implementation. This system would be able to control crop diseases in a pre-emptive and cost efficient manner and avoid the current scenario of belatedly combating largescale epidemics. Components would include: risk assessment to predict impacts on food and feed value chains; targeted surveillance; fit-for-purpose diagnostics; control intervention packages; extension mechanisms; and enabling policy environments. Each component would be refined, based on practical feedback and results from research targeted to address knowledge gaps. Specific examples are presented for viruses of cassava, viral and bacterial diseases of banana, stem rust of wheat and a new viral disease complex of maize. Finally, the links among disease control and improved crop quality, consumer health and safe trade are discussed through biological control interventions for aflatoxin in SSA.

High Potassium, Calcium, and Nitrogen Application Reduce Susceptibility to Banana Xanthomonas Wilt Caused by Xanthomonas campestris pv. musacearum

The effect of exogenous applications of potassium (K), calcium (Ca), and nitrogen (N) on the susceptibility of four banana cultivars to Banana Xanthomonas wilt (BXW) was studied. Murashige and Skoog (MS) medium with normal concentrations of K at 783 mg/liter, Ca at 121 mg/liter, and N at 841 mg/liter was modified to contain various concentrations of K, Ca, and N. Each nutrient was varied singly, each with three replicate experiments. The concentrations were K at 78, 157, 391, 783, 1,565, and 3,913 mg/liter; Ca at 12, 24, 60, 121, 241, and 603 mg/liter; and N at 84, 168, 420, 841, and 1,682 mg/liter. Plantlets were generated in vitro on normal MS medium and later exposed to the nutrient concentrations for a total of 8 weeks. Thereafter, they were artificially inoculated with Xanthomonas campestris pv. musacearum using an insulin syringe. In each nutrient, plantlets exposed to higher nutrient concentrations significantly (P < 0.0001) accumulated more nutrient in their tissues compared with those exposed to lesser nutrient concentrations. Wilt incidences were significantly reduced, and incubation periods (time from inoculation to appearance of first disease symptoms) increased, with increasing nutrient application. The study lays a background for in vivo studies aimed at management of BXW using nutrients, such as fertilizer application.

First report of atypical Xanthomonas euvesicatoria strains causing bacterial spot of tomato in Nigeria.

Bacterial spot (BS) is an important disease of tomato in Nigeria (2). Although a xanthomonad was isolated from tomato in Nigeria and characterized using phenotypic and pathogenicity tests, the bacterium was not characterized genetically to confirm the species. To determine the species associated with BS, leaves were collected in fields in northwestern Nigeria from tomato plants showing typical BS symptoms, which consisted of dark, irregular-shaped brown leaf spots that coalesced, resulting in a blighted appearance. Isolations from individual lesions were made on nutrient agar (NA). Yellow, mucoid colonies typical of Xanthomonas were isolated from 14 lesions and all were determined to be amylolytic (3). To determine the races of these strains, bacterial suspensions of the tomato strains, derived from 24-h cultures grown on NA at 28°C, were adjusted to 108 CFU/ml and infiltrated into leaves of tomato and pepper differential genotypes (5). The tomato strains elicited hypersensitive reactions (HRs) on the four pepper differential lines and an HR on the tomato genotype FL 216, which contains the R gene Xv3, but elicited susceptible reactions on the tomato genotypes Hawaii 7998 and Bonny Best. These reactions are typical of X. perforans tomato race 3 strains (5). Multilocus sequence analysis (MLSA) of six housekeeping genes (fusA, lacF, gyrB, gltA, gapA, and lepA) was used to further analyze four representative strains (1) (GenBank Accession Nos. KJ938581 to KJ938584, KJ938588 to KJ938591, KJ938595 to KJ938598, KJ938602 to KJ938605, KJ938629 to KJ938632, and KJ938636 to KJ938639, respectively). A partial sequence of hrpB2 was also made since the four Xanthomonas species associated with BS can be differentiated based on sequence divergence of this gene (3) (KJ938609 to KJ938621 and KJ938628). The housekeeping gene sequences were aligned along with other Xanthomonas sequences imported from the National Center for Biotechnology Information (NCBI) database ( www.ncbi.nlm.nih.gov ) using the MUSCLE tool from MEGA software, 5.2.2. Maximum likelihood phylogenetic trees constructed for the six housekeeping gene sequences individually and in concatenation revealed that the strains grouped most closely with the X. euvesicatoria reference strain 85-10 but more distantly to X. perforans. The hrpB2 sequence, which is highly conserved for each Xanthomonas species pathogenic on tomato (4), was sequenced from the tomato strains. These sequences were identical to the hrpB2 sequence from X. perforans strains but different from X. euvesicatoria. Although BS is common in Nigeria, to our knowledge, this represents a unique group of X. euvesicatoria strains from tomato that are identical to X. perforans based on pathogenic reactions on tomato and pepper and hrpB2 sequence identity but are more closely related to X. euvesicatoria based on the six housekeeping gene sequences. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) E. U. Opara and F. J. Odibo. J. Mol. Genet. 1:35, 2009. (3) J. B. Jones et al. Syst. Appl. Microbiol. 27:755, 2004. (4) A. Obradovic et al. Eur. J. Plant Pathol. 88:736, 2004. (5) R. E. Stall et al. Annu. Rev. Phytopathol. 47:265, 2009.

First Report of Phakopsora pachyrhizi on Soybean Causing Rust in Tanzania

Phakopsora pachyrhizi Syd. was reported on legume hosts other than soybean in Tanzania as early as 1979 (1). Soybean rust (SBR), caused by P. pachyrhizi, was first reported on soybean in Africa in Uganda in 1996 (3), and its introduction into Africa was proposed to occur through urediniospores blowing from western India to the African east coastal areas by moist northeast monsoon winds (4). The fungus rapidly spread and was reported on soybean in South Africa in 2001, in western Cameroon in 2003, and in Ghana and the Democratic Republic of the Congo in 2007 (5). A second species causing SBR on soybean, P. meibomiae, has not been reported in Africa or elsewhere, outside of the Americas. From 2012 to 2014, symptomatic leaf samples were collected in the major soybean growing areas of the Tanzanian Southern Highlands (Iringa, Mbeya, and Ruvuma regions). Symptoms of SBR included yellowing of leaves and tan sporulating lesions. These symptoms were observed at flowering through seed maturity. From fields surveyed in 2012, 2013, and 2014, SBR was observed in 5 of 14, 7 of 11, and 14 of 31 fields, respectively. Some of the leaves sampled had up to 80% of the leaf area affected. When microscopically examined, urediniospores were elliptical, echinulate, and hyaline to pale yellowish brown. In 2014, sporuliferous uredinia were observed on leaf material collected from the Iringa and Ruvuma regions of Tanzania, and a subset of these samples was sent by APHIS permit to the University of Illinois. To confirm the pathogen, symptomatic soybean leaf tissue of approximately 1 cm2 was excised from each of the samples, and DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), with further purification using the MicroElute DNA Clean-up Kit (Omega Bio-Tek, Norcross, GA). The DNA was subjected to quantitative PCR using published Taqman assays for P. pachyrhizi, P. meibomiae, and a multiplexed exogenous internal control reaction to validate negative results (2). P. pachyrhizi DNA was detected in excess of 66,000 genome equivalents/cm2 in all samples, and P. meibomiae DNA was determined to be absent from all samples (limit of quantification ~2 pg DNA/cm2). Free surviving urediniospores were dislodged from 12 samples and inoculated onto susceptible soybean cultivar Williams 82, which produced sporulating SBR lesions after 2 weeks of incubation in a detached-leaf assay. Thus, Kochs postulates were completed. This is the first report of P. pachyrhizi causing rust on soybean in Tanzania. In vivo cultures have been established from most of these samples, and ongoing research includes an evaluation of the P. pachyrizi virulence on a differential set, and characterization of the genetic diversity. References: (1) D. L. Ebbels and D. J. Allen. Phytopath. Pap. 22:1-89. (2) J. S. Haudenshield and G. L. Hartman. Plant Dis. 95:343, 2011. (3) R. Kawuki et al. Afr. Crop Sci. J. 11:301, 2003. (4) C. Levy. Plant Dis. 89:669, 2005. (5) P. S. Ojiambo et al. Plant Dis. 91:1204, 2007.

First report of Phakopsora pachyrhizi causing rust on soybean in Malawi.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, has become established in Africa since the first report in Uganda in 1996 (2). The urediniospores, as windborne propagules, have infested new regions of Africa, initiating SBR in many countries, including Ghana and Democratic Republic of the Congo in 2007 (4) and Tanzania in 2014 (3). No refereed reports have been published about rust in Malawi, but some people have indicated that soybean rust may have been observed as early as 2008. Typical symptoms and signs of SBR, including leaf yellowing and tan, sporulating uredinia, were observed on soybean in May 2014 during field surveys in the major soybean-growing areas of Malawi, including the central (Dowa, Mchinji, and Kasungu) and southern (Thyolo) regions in nine out of 12 sites surveyed. When microscopically examined, urediniospores were elliptical, echinulate, and hyaline to pale yellowish brown. Leaves exhibiting sporuliferous uredinia were sent by APHIS permit to the University of Illinois. To confirm the pathogen, symptomatic soybean leaf tissue of approximately 1 cm2 was excised from each of the samples, and DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), with further purification using the MicroElute DNA Clean-up Kit (Omega Bio-Tek, Norcross, GA). The resulting DNA was analyzed by quantitative PCR using published Taqman assays for P. pachyrhizi and P. meibomiae, with a multiplexed exogenous internal control reaction to validate negative results (1). P. pachyrhizi DNA was detected in excess of 180,000 genome equivalents/cm2 in all samples, indicating a substantial infection. P. meibomiae DNA was determined to be absent from all samples, within the limit of quantification of ~2 pg DNA/cm2. Urediniospores dislodged from three leaves and inoculated onto susceptible soybean cultivar Williams 82 produced tan lesions after 2 weeks of incubation in a detached-leaf assay. This is the first confirmed report of P. pachyrhizi causing rust on soybean in Malawi, putting at risk 14,000 ha currently under soybean production. The reports of soybean rust in Malawi and adjoining countries will alter soybean production practices and research interests. In some cases, foliar application of fungicides has increased and planting dates have been changed to avoid conditions that are most conducive for rust development. Efforts to understand the virulence and genetic diversity of the pathogen in the region are needed in order to develop and deploy resistant cultivars. References: (1) J. S. Haudenshield and G. L. Hartman. Plant Dis. 95:343, 2011. (2) R. Kawuki, et al. Afr. Crop Sci. J. 11:301, 2003. (3) H. M. Murithi et al. Plant Dis. 98:1586, 2014. (4) P. S. Ojiambo et al. Plant Dis. 91:1204, 2007.

First Report of Xanthomonas euvesicatoria Causing Bacterial Spot Disease in Pepper in Northwestern Nigeria

Bacterial spot (BS) has been reported as an important disease on pepper in Nigeria (4). Xanthomonas campestris pv. vesicatoria was identified as the causal agent using phenotypic and pathogenicity tests; however, X. campestris pv. vesicatoria is a synonym for two genetically distinct groups that have been elevated to the species X. euvesicatoria and X. vesicatoria (2). Furthermore, the latter two species and X. gardneri cause similar diseases on pepper (2). In order to determine the species associated with BS on pepper, leaves with irregular, dark brown lesions were collected from pepper plants in fields from northwestern Nigeria, and isolations were made on nutrient agar (NA). Yellow, mucoid colonies typical of Xanthomonas were isolated. Six strains isolated from pepper were determined to be non-amylolytic. For race determinations, bacterial suspensions of the pepper strains, derived from 24-h cultures grown on NA at 28°C, were adjusted to 108 CFU/ml and infiltrated into leaves of tomato and pepper differential genotypes (5). The six pepper strains elicited HRs on the tomato differential genotypes. The strains produced a susceptible reaction on all pepper differentials and were designated as pepper race 6 (5). Multilocus sequence analysis (MLSA) using six housekeeping genes (fusA, lacF, gyrB, gltA, gapA, and lepA) was used to further analyze the strains (1) (GenBank Accession Nos. KJ938585 to KJ938587, KJ938592 to KJ938594, KJ938599 to KJ938601, KJ938606 to KJ938608, KJ938633 to KJ938635, and KJ938640 to KJ938642). A partial sequence of hrpB2 was also sequenced since the four Xanthomonas species associated with BS can be differentiated based on sequence divergence (3) (KJ938622 to KJ938627). The housekeeping gene sequences were aligned along with other Xanthomonas sequences imported from the NCBI database using muscle tool from MEGA software, 5.2.2. Maximum likelihood phylogenetic trees constructed for the six housekeeping gene sequences individually and in concatenation revealed that the Nigerian pepper strains were identical to the X. euvesicatoria reference strain 85-10. Although BS is common in Nigeria, to our knowledge, this represents the first report for this pepper pathogen in Nigeria. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (3) J. B. Jones et al. System Appl. Microbiol. 27:755, 2004. (4) A. Obradovic et al. Eur. J. Plant Pathol. 88:736, 2004. (2) E. U. Opara and F. J. Odibo. J. Mol. Gen. 1:35, 2009. (5) R. E. Stall et al. Ann. Rev. Phytopathol. 47:265, 2009.

Spread of Xanthomonas campestris pv. musacearum in banana plants: implications for management of banana Xanthomonas wilt disease

Banana Xanthomonas wilt (BXW) caused by Xanthomonas campestris pv. musacearum (Xcm) is a devastating disease of bananas in Uganda and across the Great Lakes region of East and Central Africa. While use of disease-free suckers is recognized as important to control BXW, bacterial movement from infected mother plants to their suckers is not well understood. In this study, the movement of Xcm through the pseudostem of naturally and artificially infected bananas was examined. In naturally infected plants, samples of plant organs collected from susceptible cultivars ‘Kayinja’, ‘Nfuuka’ and Kivuuvu’ (Musa acuminata) at various stages of disease were analysed using a polymerase chain reaction assay employing Xcm specific primers. Xanthomonas campestris pv. musacearum was detected in 70% of asymptomatic corms and suckers collected from each of the three susceptible cultivars. In ‘Kayinja’ and ‘Nakitembe’, Xcm was recovered from plant parts 20 cm away from the point of inoculation prior to symptom development. The population of Xcm was variable within and among the plant parts over time, with the highest number being recorded in the inoculated region for all cultivars. No disease was observed seven days after inoculation of the Xcm-resistant wild species M. balbisiana and Xcm was restricted to the point of inoculation. This study implies that by the time wilt symptoms are expressed, Xcm has migrated from the point of entry to most parts of the plants. Use of suckers from infected plants should be restricted as they are likely to be latently infected and could thus result in disease when transplanted.

The need for culture collections to support plant pathogen diagnostic networks.

Plant-pathogenic microorganisms, by virtue of their size, similarity in disease symptoms and closely related morphologies, are notoriously difficult to diagnose and detect. Diagnosis gives proof as to the causal agent of disease and is important for developing appropriate control measures. Detection shows the presence of a microorganism and is of importance for safeguarding national and international trade. Live reference collections are required to characterize the taxonomy and function of microorganisms as a prerequisite to development of tools for diagnosis and detection. Two case studies will be presented in this paper to demonstrate the importance of microorganism collections for facilitating knowledge sharing and the development of identification methods. Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense and sharka disease of stone fruits caused by plum pox virus (PPV) are considered. Both diseases consist of different races/strains with different host specificities, but Fusarium wilt poses a threat to food security, while PPV poses a threat to trade due to its classification as a quarantine pest, since there is no anti-virus treatment available to control sharka disease in orchards. It is only through comprehensive collections of correctly identified and well-maintained strains representing the genetic diversity of a target organism that robust, specific, reliable and efficient diagnostic and detection tools can be developed.