Pathogenic variation of Phakopsora pachyrhizi infecting soybean in Nigeria.

Soybean rust, caused by Phakopsora pachyrhizi, is an important disease in Nigeria and many other soybean-producing countries worldwide. To determine the geographical distribution of soybean rust in Nigeria, soybean fields were surveyed in the Derived Savanna (DS), Northern Guinea Savanna (NGS), and Southern Guinea Savanna (SGS) agroecological zones in Nigeria between 2004 and 2006. Disease severity in each zone was determined and analyzed using geostatistics. Prevalence of infected fields and disease severity in surveyed fields were significantly (P < 0.05) different between geographical zones with both variables being higher in the DS zone than in either NGS or SGS zones. Geostatistical analysis indicated that the spatial influence of disease severity at one location on severity at other locations was between 75 and 120 km. An exponential model best described the relationship between semivariance and lag distance when rust severity was high. Spatial interpolation of rust severity showed that locations in the DS zone were more conducive for the rust epidemic compared to areas in the NGS zone. In the 2005 survey, 116 purified isolates were established in culture on detached soybean leaves. To establish the nature of pathogenic variation in P. pachyrhizi, a set of four soybean accessions with Rpp(1), Rpp(2), Rpp(3), and Rpp(4) resistance genes, two highly resistant and two highly susceptible genotypes were inoculated with single uredinial isolates. Principal component analysis on the number of uredinia per square centimeter of leaf tissue for 116 isolates indicated that an adequate summary of pathogenic variation was obtained using only four genotypes. Of these four, PI 459025B (with Rpp(4) gene) and TGx 1485-1D had the lowest and highest number of uredinia per square centimeter, respectively. Based on cluster analysis of the number of uredinia per square centimeter, seven pathotype clusters were determined. Isolates in cluster III were the most virulent, while those in cluster IV were the least virulent. Shannon's index (H) revealed a more diverse pathogen population in the DS zone (H = 1.21) compared to the rust population in SGS and NGS with H values of 1.08 and 0.91, respectively. This work will be useful in breeding and management of soybean rust by facilitating identification of resistant genotypes and targeting cultivars with specific resistance to match prevailing P. pachyrhizi pathotypes in a given geographical zone.

Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize.

Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic and a public health concern. Therefore, strategies for controlling aflatoxin contamination in maize are being investigated. The abilities of eleven naturally occurring atoxigenic isolates in Nigeria to reduce aflatoxin contamination in maize were evaluated in grain competition experiments and in field studies during the 2005 and 2006 growing seasons. Treatments consisted of inoculation of either grains in vials or ears at mid-silking stage in field plots, with the toxigenic isolate (La3228) or atoxigenic isolate alone and co-inoculation of each atoxigenic isolate and La3328. Aflatoxin B(1) + B(2) concentrations were significantly (p < 0.05) lower in the co-inoculation treatments compared with the treatment in which the aflatoxin-producing isolate La3228 was inoculated alone. Relative levels of aflatoxin B(1) + B(2) reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B(1) + B(2) concentrations in both laboratory and field trials. These two isolates have potential value as agents for the biocontrol of aflatoxin contamination in maize. Because these isolates are endemic to West Africa, they are both more likely than introduced isolates to be well adapted to West African environments and to meet regulatory concerns over their use throughout that region.

Distribution and toxigenicity of Aspergillus species isolated from maize kernels from three agro-ecological zones in Nigeria.

Maize samples were collected during a survey in three agro-ecological zones in Nigeria to determine the distribution and aflatoxin-producing potential of members of Aspergillus section Flavi. The three agro-ecological zones were, Derived Savannah (DS) and Southern Guinea Savannah (SGS) in the humid south and North Guinea Savannah (NGS) in the drier north. Across agro-ecological zones, Aspergillus was the most predominant fungal genera identified followed by Fusarium with mean incidences of 70 and 24%, respectively. Among Aspergillus, A. flavus was the most predominant and L-strains constituted >90% of the species identified, while the frequency of the unnamed taxon S(BG) was <3%. The incidence of atoxigenic strains of A. flavus was higher in all the districts surveyed except in the Ogbomosho and Mokwa districts in DS and SGS zones, respectively, where frequency of toxigenic strains were significantly (P<0.05) higher than that of atoxigenic strains. The highest and lowest incidence of aflatoxin positive samples was recorded in the SGS (72%) and NGS (20%), respectively. Aflatoxin contamination in grain also followed a similar trend and the highest mean levels of B-aflatoxins were detected in maize samples obtained from Bida (612 ng g(-1)) and Mokwa (169 ng g(-1)) districts, respectively, in the SGS. Similarly, the highest concentrations of G-aflatoxins were detected in samples from Akwanga district in the SGS with a mean of 193 and 60 ng g(-1), respectively. When agro-ecological zones were compared, B-aflatoxins were significantly (P<0.05) higher in SGS than in NGS, and intermediate in maize samples from the DS agro-ecological zone.

Evaluation of Soybean Germplasm for Resistance to Soybean Rust (Phakopsora pachyrhizi) in Nigeria.

Soybean rust, caused by Phakopsora pachyrhizi, is one of the most important constraints to soybean production worldwide. The absence of high levels of host resistance to the pathogen has necessitated the continued search and identification of sources of resistance. In one set of experiments, 178 soybean breeding lines from the International Institute of Tropical Agriculture were rated for rust severity in the field in 2002 and 2003 at Ile-Ife, Yandev, and Ibadan, Nigeria. Thirty-six lines with disease severity ≤3 (based on a 0-to-5 scale) were selected for a second round of evaluation in 2004 at Ibadan. In the third round of evaluation under inoculated field conditions, 11 breeding lines with disease severity ≤2 were further evaluated for rust resistance at Ibadan in 2005 and 2006. The breeding lines TGx 1835-10E, TGx 1895-50F, and TGx 1903-3F consistently had the lowest level of disease severity across years and locations. In another set of experiments, 101 accessions from the United States Department of Agriculture-Agricultural Research Service and National Agriculture Research Organization (Uganda) were evaluated in the first round in 2005 under inoculated conditions in the screenhouse; 12 accessions with disease severity ≤20% leaf area infected were selected for evaluation in the second round in 2005 and 2006 under inoculated field conditions at Ibadan. Highly significant differences (P < 0.0001) in disease severity were observed among the 101 accessions during this first round of rust evaluation. Significant (P < 0.0001) differences in rust severity and sporulation also were observed among the 12 selected accessions. Accessions PI 594538A, PI 417089A, and UG-5 had significantly (P < 0.05) lower disease severity than all other selected accessions in both years of evaluation, with rust severities ranging from 0.1 to 2.4%. These results indicate that some of the breeding lines (TGx 1835-10E, TGx 1895-50F, and TGx 1903-3F) and accessions (PI 594538A, PI 417089A, and UG-5) would be useful sources of soybean rust resistance genes for incorporation into high-yielding and adapted cultivars.

Rapid Screening of Musa Species for Resistance to Black Leaf Streak Using In Vitro Plantlets in Tubes and Detached Leaves.

This study investigated the utility of inoculation of in vitro plantlets in tubes and detached leaves as reliable and rapid assays for screening Musa genotypes against Mycosphaerella fijiensis, the causal agent of black leaf streak. In the first part of the study, three types of inocula were evaluated to determine suitability for in vitro inoculation. Inoculation of in vitro plantlets with mycelial fragments resulted in significantly (P < 0.05) higher levels of disease severity and faster rates of disease progress compared with inoculations using conidial suspensions. In the detached leaf assay, amending agar medium with plant hormones significantly (P < 0.0001) aided retention of green leaf color. Leaf pieces on medium containing gibberellic acid at 5 mg/liter had about 5% chlorosis at 52 days after plating. When in vitro plantlets in tubes and detached leaves of 10 Musa genotypes with different levels of disease resistance were inoculated with M. fijiensis, there were significant (P < 0.05) differences among genotypes in leaf area infected, incubation time, and symptom evolution time. For incubation time and leaf area infected, cultivars responded depending on their level of disease resistance, with resistant genotypes Calcutta-4 and PITA-17 having significantly (P = 0.001) longer incubation times and lower infected leaf areas compared with the susceptible cultivar Agbagba and moderately resistant cultivar FHIA-23. A similar pattern in cultivar response was observed for symptom evolution time. Leaf area infected was not significantly (P = 0.2817 for two-tailed t test) different when assessed using the two assays, and infected leaf areas in both assays were strongly correlated (r = 0.88, n = 48, P < 0.0001). Although incubation times were significantly (P = 0.0062 for two-tailed t test) different between the two assays, values from the two assays were strongly correlated (r = 0.69, n = 48, P < 0.0001). These results show that these two assays are rapid and space-effective, and can reliably be used for screening Musa genotypes for resistance to black leaf streak.

Comparison of Field, Greenhouse, and Detached-Leaf Evaluations of Soybean Germplasm for Resistance to Phakopsora pachyrhizi.

Fourteen soybean accessions and breeding lines were evaluated for resistance to soybean rust caused by the fungus Phakopsora pachyrhizi. Evaluations were conducted in replicated experiments in growth chambers using detached leaves and under greenhouse and field conditions. In growth-chamber experiments, inoculation of detached leaves with 1 × 106 spores/ml resulted in a significantly (P < 0.0001) higher total number of pustules and spores per unit leaf area than inoculations with lower spore concentrations. Amending agar medium with plant hormones significantly (P < 0.0001) aided retention of green leaf color in detached leaves. Leaf pieces on a medium containing kinetin at 10 mg/liter had 5% chlorosis at 18 days after plating compared with leaf pieces on media amended with all other plant hormones, which had higher levels of chlorosis. Leaf age significantly affected number of pustules (P = 0.0146) and number of spores per pustule (P = 0.0088), and 3- to 4-week-old leaves had a higher number of pustules and number of spores per pustule compared with leaves that were either 1 to 2 or 5 to 6 weeks old. In detached-leaf and greenhouse screening, plants were evaluated for days to lesion appearance, days to pustule formation, days to pustule eruption, lesion number, lesion diameter, lesion type, number of pustules, and spores per pustule in 1-cm2 leaf area. Plants also were evaluated for diseased leaf area (in greenhouse and field screening) and sporulation (in field screening) at growth stage R6. There were significant (P < 0.0001) differences among genotypes in their response to P. pachyrhizi infection in the detached-leaf, greenhouse, and field evaluations. Accessions PI 594538A, PI 417089A, and UG-5 had very low levels of disease compared with the susceptible checks and all other genotypes. Detached-leaf, greenhouse, and field results were comparable, and there were significant correlations between detached-leaf and greenhouse (absolute r = 0.79; P < 0.0001) and between detached-leaf and field resistance (absolute r = 0.83; P < 0.0001) across genotypes. The overall results show the utility of detached-leaf assay for screening soybean for rust resistance.

Effect of temperature on the survival and infectivity of Pseudotheraptus devastans vector.

The aim of this study was to investigate under a controlled environment, the effect of temperature on the survival and infectivity of Pseudotheraptus devastans Distant, a cassava anthracnose disease vector. The insect P. devastans was collected from young cassava (Manihot esculenta Crantz) field plots, at the International Institute of Tropical Agriculture, (IITA), Ibadan, Nigeria. A mixture of the different developmental stages of eggs, first to fifth instar nymphs, and adults, were incubated in controlled environment chambers, under various constant temperatures of: 15, 17, 22, 25, 27, 30, and 35 degrees C. Relative humidity at different temperature conditions were recorded and maintained at 90%, 85%, 80%, 75%, 70%, 65%, and 60%, respectively. A significant increase in insect survival was observed between 22 and 27 degrees C temperature conditions while a significant decrease in survival was observed at 15 degrees C and above 30 degrees C. Lesion number, lesion diameter and infectivity among the insect stages varied as a function of temperature and relative humidity. Infectivity was highest at 22-25 degrees C maintained at 75-80% RH and lowest at 15 degrees C and above 30 degrees C maintained respectively, at 65% RH and 90% RH. There was considerable low vector infectivity due to low survival of the insects at extreme temperatures.

In vitro, greenhouse and field assessments of cassava lines for resistance to anthracnose disease caused by Colletotrichum gloeosporioides f.sp. manihotis.

Fifty-three cassava lines were selected from breeding populations at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria and screened in vitro for resistance to cassava anthracnose disease (CAD). The in vitro inoculation of stem cuttings with the fungus Colletotrichum gloeosporioides f.sp. manihotis showed significant differences (p +/- 0.05) in acervuli production and in the sensitivity of the cassava lines to the fungal infection after 7 days of incubation at 25 degrees C. Cassava lines 88/01084, 91/00595, 91/00475, 91/00344, 91/00684, 91/00313, 91/00422, and 91/00344 were highly resistant, with necrotic lesion sizes less than 7 mm. In contrast pedigree lines 88/02549, 89/0008, 91/00390 and 91/00402 were highly susceptible with the largest necrotic lesion size being greater than 20 mm. Ten cassava lines from the in vitro screening that showed varying levels of resistance to CAD were selected, based on their flowering abilities for diallel hydridization trials, and were further screened in greenhouse and field trials for CAD resistance. The greenhouse and field screening showed significant varietal differences (p +/- 0.05) in sensitivity to the fungus. In all cases, the progeny lines showed correlated levels of resistance irrespective of the type of screening or assessments. Correlation analysis of the in vitro, greenhouse and field assessments showed that there was a good correspondence among all three methods of evaluating for CAD.

Identification and survival of the causal organism of leaf smut disease of cowpea in Nigeria.

Protomycopsis phaseoli (Ramak and Subram) is the causal agent of the cowpea leaf smut disease in Nigeria and not Entyloma vignae as claimed by some authors. This pathogen formed dark ash-grey to sooty-black lesions of 3-10 mm in diameter, while young lesions had yellow haloes. P. phaseoli produced dark reddish-brown chlamydospores that are globose to oval measured 23.8 microm, thick-walled and rugose. The chlamydospores germinated and produced globose vesicles. The pathogen grew on potato dextrose agar only when the leaf tissue was dipped in acidified water (1% H2SO4). The organism was slowly growing at 24-28 degrees C with snow white colour. Chlamydospores of P. phaseoli in infected cowpea leaves survived longer when buried in the soil for five months than when they were left on the soil surface for the same period at temperatures (26-27 degrees C) and humidity (70-82%) prevailing in Ibadan. Destruction of leaf debris before crop emergence, long period of rotation and no tillage cropping are suggested to prevent the onset and spread of leaf smut disease of cowpea.

Evaluation of ash from some tropical plants of Nigeria for the control of Sclerotium rolfsii Sacc. on wheat (Triticum aestivum L.).

Eleven ash samples, from organs of nine tropical plants, were screened for their abilities to inhibit mycelial growth and sclerotial germination of a Nigerian isolate of Sclerotium rolfsii on agar and in the soil. Ten ash samples showed some activity against mycelial growth of S. rolfsii in vitro. Ash samples from Delonix regia stem wood, Mangifera indica leaf and Vernonia amygdalina leaf were most effective as each totally inhibited mycelial growth of S. rolfsii in vitro. Ocimum gratissimum leaf ash, D. regia wood ash and Musa paradisiaca flower bract ash inhibited sclerotial germination on agar. Nine ash samples protected seeds against pre-emergence rot. Ash from M. indica leaf, V. amygdalina leaf and Azadirachta indica leaf protected seedlings against post-emergence infection. Eichornia crassipes ash, which was ineffective in vitro, offered some protection to seeds in soil against pre-emergence rot. The study demonstrates potentials of ash samples from tropical plants in control of S. rolfsii on wheat.

First Report of Colletotrichum gloeosporioides f. sp. manihotis, Cause of Cassava Anthracnose Disease, Being Seed-borne and Seed-Transmitted in Cassava.

Cassava anthracnose disease is a major economic disease of cassava in the tropics (2). Infection can lead to a significant loss in planting materials and total field crop failure. The disease has been reported to be transmitted mainly by a bug (Pseudotheraptus devastans Dist) (1). Open pollinated seeds from 13 cassava (Manihot esculenta Crantz) genotypes, stored for 10 months after harvest in 1994, were used to investigate the presence of the cassava anthracnose disease fungus. Seeds, 200 from each genotype, were surface sterilized, cultured on potato dextrose agar (PDA), and incubated for 8 days, at 25 ± 2°C. Microscopic examination indicated that Colletotrichum gloeosporioides was one of the seed-borne fungi, with up to 40% incidence recorded in some genotypes. Seeds from five susceptible genotypes selected for seed transmission studies were planted in fine, steam-sterilized soils in jiffy pots and watered daily for seedling emergence. At a height of 10 to 15 cm, the seedlings were transferred to plastic pots (10.5 cm in diameter) filled with sterilized mixture of soil and sand (2:2, vol/vol). Pots were placed close to each other to obtain a thick plant canopy. Temperature of 25 to 32°C and humidity of 80 to 98% were maintained. After 45 days, some plants had cassava anthracnose symptoms, including defoliation, wilt, and necrotic lesions. Stems, leaves, and roots of infected plants were washed, surface sterilized, and plated on PDA for 5 to 7 days. Microscopic observation of the fungus showed conidia of C. gloeosporioides. The rest of the plants were monitored for 3 months under vector-free conditions for typical anthracnose symptoms. Mean maximum wilt and defoliation of 35 to 38% was recorded in some genotypes. Conidial suspensions of C. gloeosporioides were used in stem-puncture inoculations of young, healthy cassava plants. The typical anthracnose symptoms of stem necrosis were observed 2 weeks after inoculation, confirming isolates as C. gloeosporioides f. sp. manihotis. This is the first report of C. gloeosporioides f. sp. manihotis being seed-borne and seed-transmitted in cassava. References: (1) B. Boher et al. Agronomie 3:989, 1983. (2) J. C. Lozano. PANS 20:30, 1974.

The fate of aflatoxins during the production of "Ogiri", a West African fermented melon seed condiment from artificially contaminated seeds.

Twenty-six market samples and four laboratory-prepared samples of "ogiri" were screened for aflatoxin contamination. Aflatoxins were not detected in any of the samples. The fermented product (ogiri) was prepared with Aspergillus flavus-contaminated melon seeds. Losses of 64.7% aflatoxin B1 and 82.9% aflatoxin G1 were observed at the end of the third day of fermentation of the ground melon seeds. The samples were completely detoxified at the fourth day of fermentation. Increase in pH of the mash from 6.2 to 7.2 was observed during fermentation.

Reduction of aflatoxin content of infected cowpea seeds during processing into food.

Cowpeas have been a major source of protein, especially in developing nations. Despite of the fact that aflatoxin contaminations have been found to be mainly common in substrates such as carbohydrates, detection of the toxins in raw cowpeas has also been reported [7]. It is therefore important and necessary to investigate fate of the aflatoxin when raw cowpeas are processed into various products. In Nigeria, cowpea seeds are usually consumed after boiling to softness and mixed with ingredients such as pepper, salt and palm-oil to form a porridge. Other processing methods involve wet milling the cowpea seeds and either steaming in aluminium cups or in leaves to form 'moinmoin', or frying in oil to form 'akara' balls. In this work therefore, the fate of the aflatoxins as a result of the processing of artificially contaminated cowpeas was investigated.

Microorganisms associated with mouldiness of dried yam chips and their prevention.

The broad objective of this study was to isolate and identify the microorganisms causing mouldiness of stored yam chips and to look for ways of preventing the problem. Microorganisms isolated included Aspergillus flavus, A. glaucus, A. nidulans, A. niger, A. ochraceous, A. tamarii, A. candidus, Penicillium oxalicum, Trichoderma longibrachyatum, Rhizopus nigricans, Cylindrocarpon radicicola, Neurospora crassa, Botryodiplodia theobromae, Bacillus subtilis, Bacillus cereus, Erwinia carotovora and Serratia marcescens. Some of these microorganisms are transient invaders. Of the calcium-based chemicals used to prevent mouldiness, only CaCO3 and Ca(OH)2 proved effective and also prevented infestation of the chips by storage pests throughout the period of study. Whereas the product of the untreated yam chips was preferred in terms of colour, the product of CaCO3- and Ca(OH)2-treated yam chips were preferred in terms of taste and texture. As for physiological reaction after eating the products of the treated chips, Ca(OH)2- treated chips seemed to be more preferred by the members of the tasting panel.

Isolation, purification and assay of the macerating enzyme produced by Penicillium oxalicum Curie and Thom.

Penicillium oxalicum produced two isozymes of polygalacturonase (PG) and a pectate lyase (PL). The enzymes were separated and purified following ammonium sulphate precipitation, ion exchange chromatography, ultrogel column chromatography and isoelectric focusing. The first isozyme of polygalacturonase (PGI) was rather unstable hence its properties could not be much assayed. PGII macerated and killed yam tissue in 4 hours but PL was unable to do so. Enzyme assay for the end-products of degradation of sodium polypectate and yam tissue showed that PGI was an exo-enzyme while PGII and PL were endo-enzymes. Endo-polygalacturonase (PGII) appears to play the major role (as the macerating enzyme) in the pathogenesis of yam tissue infected by P. oxalicum.

Production of oxalic acid by some fungi infected tubers.

Oxalic acid (as oxalate) was detected in four tubers commonly used for food in Nigeria-Dioscorea rotundata (White yam), Solanum tuberosum (Irish potato), Ipomoea batatas (Sweet potato), and Manihot esculenta (cassava). Whereas healthy I. batata had the highest oxalic acid content, healthy M. esculenta contained the lowest. When all tubers were artifically inoculated with four fungi-Penicillium oxalicum CURIE and THOM, Aspergillus niger VAN TIEGH, A. flavus and A. tamarii KITA, there was an increase in oxalate content/g of tuber tissue. The greatest amount of oxalate was produced by P. oxalicum in D. rotundata tuber. Consistently higher amounts of oxalate were produced by the four fungi in infected sweet potato tuber than in any other tuber and consistently lower amounts of oxalate were produced by the four fungi in Irish potato tuber. Differences in the carbohydrate type present in the tubers and in the biosynthesis pathway are thought to be responsible for variation in the production of oxalate in the different tubers by the four fungi used.