Determining the epidemiological value of resistance or tolerance to principle soilborne sorghum and soybean diseases in South Africa

Research Outputs:

A MSc study linked to this project was completed and data were presented at the local 2013 SASPP congress and the ICPP congress in China. Chemotypes within the Fusarium graminearum species complex were evaluated and the NIV chemotype was found exclusively on sorghum as opposed to the 15A-DON chemotype that occurred exclusively on maize. Species associate with the former were F. meridionale and F. acacia-mearnsii and F. boothii with the latter. Despite the exclusive colonization of grain by the respective species, DON and NIV co-occurred on grain which indicate that mycotoxins may be translocated from other tissues eg. Stems and roots.


Field screening of sorghum cultivars for disease resistance and yield loss.

Sorghum hybrids from the National Cultivar Trials were planted at Cedara for disease evaluation, changes in host physiology and yield loss assessment. Trials were planted in a randomized split plot design with cultivars as whole plot factor and fungicide sprayed (Punch Xtra at 750 ml/ha applied at six and nine weeks after planting) and unsprayed as sub-plot factors. Leaf blight infection was relatively low and although cultivars differed in infection levels, these were not regarded as above yield reduction threshold. Grain mass (total of 10 heads) increased with fungicide sprays and gains differed with cultivar. Highest response was with CAP1003 with 9.5% yield gain compared with 0.08 % in PAN8911.

The hypothesis was that fungicides reduce endophytic fungal activity and premature closing of the grain hilum which may result in enhanced plant growth and grain density despite the absence of severe disease levels. However, ergosterol evaluation of internodes did not provide an indication of this and will be retested. In a smaller unreplicated evaluation at Ukulima (Alma) 10 row plots of PAN8420 and PAN8706(W) were sprayed with Punch Xtra 6 weeks after planting and compared with unsprayed blocks. No significant differences in yield were recorded according to Student’s t-test. This observation was attributed to the warmer and dryer conditions that reduced yield response to fungicide applications.

It is proposed that multi-location trials be initiated to quantify fungicide x environment interactions so as to critically assess conditions that will enhance plant growth. No significant effect on grain mold rating was recorded.

Evaluation of sorghum nurseries for general adaptation, yield and disease resistance

Disease evaluation of germplasm from a range of sources is on-going and is conducted in collaboration with Texas-Agrilife and the Buffet Foundation. Field trials, including 15 diverse nurseries from Texas Agrilife, Zambia and South Africa, were evaluated for adaptation to Ukulima conditions, yield potential and disease development. Sorghum nurseries included the Grain Characteristics Nursery, All Disease and Insect Nursery, Sugarcane Aphid Yield Nursery, Southern African Breeding Nursery, Drought Line Test, UFS Selection Nursery, National Cultivar Trials and Zambian Selection Nursery.

Screening included leaf blight (Exserohilum turcicum), Sooty stripe ( Ramulaspora sorghi), root rots (complex of pathogens) grain molds (complex of pathogens) and ergot (Claviceps africana). Nurseries data still requires analysis and the determination of final yield data as well as ergosterol and toxin analyses. These data will be used to select germplasm for a sub-set of nurseries for further evaluation of their potential in local farming systems. Disease development in the nurseries was limited and only low levels of leaf blight were observed.

Grain mold was the only prevalent disease and grain mold field ratings ranged from 1 to 4.5 with Dorado* Tegameo crosses giving better visual grain mold ratings.

These ratings will also serve as an important selection criterion.

Evaluation the rotation of sorghum with legumes to improve soil fertility and thus yield of sorghum as well as effects on root health and grain molds

This study was initiated in collaboration with Texas Agri-life and Buffet Foundation. Blocks of cowpea, dry bean and soybean were planted and a fallow area was left in order to evaluate the potential of legumes to provide nitrogen and other nutrients for sorghum production, especially in low input agricultural systems. The effect of rotations with these crops will commence evaluation in the 2013/14 season. Soil analyses were conducted before planting of the legumes in order to set a base line and will be repeated prior to the planting of each rotation combination.

Emphasis will be on genotype responses to changes in soil nutrients and root development and health will be monitored. Rotations will also be used to study the effect on colonization of root by mycotoxigenic fungi and the testing of the hypothesis of mycotoxin translocation from roots to grains.

Emphasis is on Fusarium thapsinum, F. verticillioides and the F. graminearum species complex and colonization is being monitored using RT-PCR. Mycotoxin translocation in tissues is being monitored using LC-MS/MS.

Potential of Fusarium oxysporum isolates to cause root rot of sorghum

Fusarium oxysporum is the most commonly isolated fungus from sorghum roots and the role in root health remains a topic of dispute. Sixty cultures from sorghum collected at a range of localities, representing colony and growth variation with the Fusarium oxysporum complex, were selected for pathogenicity tests on two cultivars, PAN8420 and PAN8706W in the greenhouse.

Root rot severity was assessed as the percentage root discolouration and ranged from 21.83-37.56% in PAN8420 and 15.26-30.05% in PAN8706W. Both cultivars showed root rot symptoms when infected with Fusarium oxysporum, but analyses of variance (P<0.05) indicated that isolates did not differ significantly from one another. The evaluation of root rot in the current study was to some extent confounded by the high level of visible root discoloration in control pots.

However, when ergosterol analyses were considered, control treatments yielded the lowest ergosterol content in roots and thus questioned the reliability of root discolouration as a root rot evaluation criterion. Ergosterol, extracted as an indicator of fungal colonization of roots, ranged from 0.00011-0.00637 µg/g in PAN8420 and 0.00008-0.00465 µg/g in PAN8706W. Fungal infection of sorghum and stress elicit the production of 3-deoxyanthocyanidins, (anthocyanin-related compounds) which serve as phytoalexins.

These compounds are responsible for the pigmentations of infected tissues in sorghum. Hence the question needs to be raised as to whether the measured response to pathogens in the current study using root discolouration, is a suitable measure of pathogenicity and thus colonization of roots or whether the observed discolourations are an indication of host resistance response to infection by the specific isolates. Furthermore, the reduced response to F. oxysporum in PAN8706(W), a low phenol, tan host would suggest that other resistance factors may be playing a role (eg. PR-proteins) and these are being evaluated to determine their exploitation potential. Egosterol is an indicator of fungal biomass and has been used successfully in the quantification of sorghum grain by grain mold pathogens. The significant differences recorded with ergosterol as an indicator of root colonization, does in fact indicate significant (P<0.05) differences in the pathogenicity of F. oxysporum isolates to colonize roots, which is contrary to the conclusion when recording pathogenicity as a factor of root discolouration.

Similarly, the significant Isolate x Cultivar interaction with ergosterol further supports the notion of differential colonization of roots. These results also indicated that some isolates were able to colonize the root tissue effectively but without producing visible symptoms on the roots or causing growth reductions. DNA sequences of the translation elongation factor 1-alpha confirmed that isolates from roots collected across a large geographical area mostly grouped in the Fusarium oxysporum species complex. The isolates in the F. oxysporum complex largely formed two phylogenetic groups that were genetically diverse.

However, none of the differences in DNA sequence were statistically well supported and most likely are indicative of a diverse genetic population infecting sorghum. More studies will be needed to determine if these groupings are linked to pathological and ecological traits.

Fusarium graminearum mycotoxins associated with grain mould of maize and sorghum in South Africa

A MSc study by Ms Mudzuli Mavhunga was completed . The study is summarized as follows:

Maize and sorghum are important crops in South Africa, meeting the dietary needs of millions of people as well as the needs of the feed industry. Fungi within the Fusarium graminearum species complex (FGSC) have recently become the subject of importance locally.

Maize kernels and sorghum grain were collected from the National Cultivar Trial over three and two seasons respectively. A total of 558 samples comprising of six maize and seven sorghum cultivars were collected from 34 and 22 localities, respectively. Although different conventional detection, isolation and identification methods were utilized, very low numbers of FGSC isolates could be obtained from the grain.

Molecular identification was based on the translation elongation factor-1a (TEF1-a) and the ammonia ligase (URA) genes. F. boothii and F. graminearum s.s. were identified from maize kernels while F. acacia-mearnsii, F. cortaderiae and F. meridionale where associated with sorghum. Results indicated host-specificity between members of the FGSC.

Total genomic DNA was extracted from all milled kernel and grain samples and analysed for the presence or absence of FGSC DNA using TaqMan real-time PCR. F. graminearum s.l. DNA was detected and quantified in over 47% of the samples.

These findings contradict previous reports that stated that this group of fungi is of minor importance. Reasons may be improved detection techniques as well as changes in agronomic practices, such as rotation of maize with wheat in South Africa, or to other shifts in environmental factors.

DON and ZEA concentrations were quantified using CD-ELISA. In the absence of an ELISA test for NIV, a selected number of samples were used in LC-MS/MS based detection and DON, NIV and ZEA were quantified. Concentrations varied considerably and no correlation between the two techniques was found. Based on CD-ELISA, maize and sorghum contamination with DON and ZEA differed significantly between seasons, localities and in some instances cultivars. An analysis of variance across all three seasons showed significant crop by locality interactions.

In maize, high levels of DON were detected in the 2007/08 season, in particular at Delmas, while during the same season ZEA contamination of maize was significantly higher at Bothaville. In sorghum, DON and ZEA contamination was highest during the 2008/09 season, although both toxins were detected in the preceding season. In both the 2007/08 and 2008/09 seasons, Cedara was the most conducive locality for grain contamination with both toxins.

Trichothecene chemotyping was conducted using both simplex and multiplex PCR. All the F. boothii and F. graminearum s.s. isolates from maize were DON/15-acetyldeoxynivalenol (15-ADON) producers and F. acacia-mearnsii, F. cortaderiae and F. meridionale isolates from sorghum grain produced NIV. DON can act as a virulence factor in plant disease and is usually associated with greater pathogenicity on plants than NIV producers.

On the other hand, NIV is believed to be more toxigenic to both humans and animals. Isolates of the 15-ADON chemotype are reportedly less toxic that those of the 3-ADON chemotype. The 3-ADON chemotype was not recorded in this study. However, more FGSC isolates need to be collected from South African cereal grains and evaluated for their mycotoxin potential to establish safety guidelines for end users of these products.

The results of this study showed that levels of field infection of maize and sorghum grain by the FGSC are of significance. Future studies need to quantify the relationships between members of the FGSC and their tricthothecene and ZEA production in South African maize and sorghum production systems including the role of cultivar choice, weather, rotation, tillage and other practices with the aim of establishing intervention technologies.

Effect of cultivar and environment on grain mold severity and mycotoxin accumulation in sorghum

Grain mold pathogens of sorghum cause reductions in grain quality and may produce mycotoxins which are potentially harmful to animals and humans. Single spore isolations of fungi isolated from surface sterilized grain on ½ Malt Extract Agar and Rose-Bengal Medium were identified using DNA sequencing based on the ITS4, ITS5, Ef1 and Ef2 primer sets.

Members of the Fusarium graminearum species complex and Phoma sorghina were isolated most frequently. Grain from nine sorghum cultivars, harvested over three seasons, was evaluated for disease on the threshed grains as an indication to disease in the field (0-5 scale), ergosterol content, total grain sugar content and the mycotoxins, deoxynivalenol, nivalenol, zearalenone. Cultivars differed significantly in threshed grain disease ratings and ergosterol content but these values were not correlated (R2 = 0.46; P>0.05). Ergosterol was regarded as the more reliable criterion since it measures total grain fungal biomass while threshed grain disease ratings can be confounded by superficial colonization. Additive Main

Effect and Multiplicative InteractionAnalysis (AMMI) of ergosterol content indicated a relatively stable response in some cultivars to changing environmental conditions with IPCA1 scores < 1. Higher IPCA1 scores for PAN8806, NS5655, PAN8816 and PAN8706(W) indicated significant GxE interactions. Respective effects of cultivar, environment and GxE interaction were 61, 24 and 15 %. Similar GxE responses were recorded for total grain sugar content and the accumulation of mycotoxins.

Results indicate the importance of including environmental variation in the screening and selection for resistance in sorghum genotypes to sorghum grain molds.

A 22 entry Sorghum Grain Characteristics Nursery, sourced from Texas A&M University has been planted on two planting dates at Cedara since 2009/10. Variations in germplasm include the presence or absence of a testa, high and low phenol contents, red, tan and purple plant/glume colour and white, red and brown grain colour.

The goal of the study is to determine physiological markers that may be used as a selection criterion for disease resistance since field ratings, mycotoxin levels and actual colonization are poorly correlated. In addition, florets, soft dough grain were collected during the season and have been frozen until all plant material has been gathered after which analyses including phenols/flavanoids, and anti-fungal proteins will be conducted. Analyses for ergosterol (as a measure of active fungal colonization) contents were conducted and were compared with visual field ratings.

Mycotoxin levels in the samples will be analysed with emphasis on ZEA, DON and NIV, toxins known to occur at significant levels on sorghum grain. Seasonal variation was analyzed using AMMI analysis to determine the stability of the measured genotypic responses. Field ratings ranged from 1.91 in TX 635 to 4.08 in Dobbs. Ergosterol content in grain ranged from 325.4 µg/g in TAM 428 to 2150 µg/g in TX 2197. There was no relationship between field rating and ergosterol content (R²=0.003) supporting the view that estimates of grain colonization using field rating can be confounded by superficial colonization. AMMI analysis of field rating indicated a relatively stable response in lines to changing environments with all IPCA1 scores <1. Respective effects of genotype, environment and GxE interaction were 44.33, 21.10 and 34.57 %.

Less stable responses were recorded for ergosterol with IPCA scores exceeding 1 in many lines. Respective effects of genotype, environment and GxE interaction were 59.61, 12.55, and 27.83 %. The relationship between grain mold severity and AFP’s and grain phenolic content needs to be determined.

Stability of mycotoxins under processing conditions

This component of the study was initiated in March 2013 in collaboration with Texas Agri-life and Buffet Foundation. This study will include the assessment of Fusarium graminearum species complex in grain as well as processing components such as bran (effect of decortication) and final products using RT-PCR. Mycotoxin levels in the various components will also be assessed.

The study will link up with a local, major processing company and will also link in with Prof. John Taylor’s component of the Ukulima study. Sample collection from the processing plant commenced in March 2013 and will continue at three-monthly intervals until September 2014. A breakdown in the LC-MS/MS (subsequently repaired) has delayed analysis of the initial samples.

Local and International Congresses

Van Rooyen, D. & McLaren, N.W. (2013) Fungi associated with root rot of sorghum in South Africa. Proceedings of the 48th Annual Plant Pathology Congress, 20-24 January, 2013. ATKV-Klein Kariba, Bella Bella.

Coetzee, L. & McLaren, N.W. (2013). Effect of cultivar and environment on on grain mold severity and mycotoxin accumulation in sorghum. Proceedings of the 48th Annual Plant Pathology Congress, 20-24 January, 2013. ATKV-Klein Kariba, Bella Bella.

Mavhunga, M., Koch, S., Flett, B.C., McLaren, N.W. (2012). Fusarium graminearum (sensu lato) mycotoxins associated with grain mold of maize and sorghum in South Africa. International MPU Workshop. Plant Protection for the Quality and safety of the Mediteranean Diet. 24-26 October, Bari, Italy.

Mavhunga, M., Koch, S., Flett, B.C., McLaren, N.W. (2012). Fusarium graminearum mycotoxins associated with grain mold of maize and sorghum in South Africa. 10th International Congress of Plant Pathology, Beijing, China, 25-30 August 2013. Poster presentation. Acta Phytopathologica Sinica 43, p 361.

McLaren, N.W. Van Rooyen, D. Coetzee, L.A (2013) Variation in sorghum grain mold and mycotoxins due to host genotype and environment. ISM-MycoRed International Conference Europe 2013. Global Mycotoxin Reduction Strategies. 27-31 May, 2013. Martina Franca, Apulia, Italy.