Continuation Research Projects

1. Studies on the use of bio-control agents on groundnut to control Aspergillus spp. and other soil pathogens

   Prof M Laing
   University of KwaZulu-Natal

   • Only one groundnut variety (tofu) was planted in two different sites (Ukulinga and Baynesfield) because we did not get enough land especially in Baynesfield.
   • Seedling transplants was successful with 100% survival at both sites compared to planting seeds.
   • Foliar diseases (Cercospora and Altenaria) were apparent at both sites, and were controlled by spraying with (mancozeb and SCORE).
   • Weeds, especially crabgrass (Diaitaria sanauinolis) were a problem at both sites and we dug it out because it has very strong roots. A herbicide (Basagran®) was sprayed to control broad leaves.
   • Pods were harvested and then left in a drying room to dry before processing.
   • Currently we are in the process of shelling. We are using hand shelling. We do not have shelling machine designed for groundnut. We will investigate buying or building a small Sheller.

   

   Data collected so far

   • Strain WB74 and G633I1 rhizobium strains showed better yield compared to control and the rest of the other strains.
   • There was no significant difference between the different rhizobium strains in the number of pink nodules.
   • Strains (Ukulinga, SCI and S29) resulted in increases in biomass compared to control and the rest of the other strains.

   Table 1: The effect of Rhizobia on number of nodules, dry weight and yield

   Rhizobia strains	Yield (g/plot)		Pink Nodules %		Biomass (g/plant)
   93015	(0.87) 0.79	a	92.80	a	37.4	abed
   Controls	(0.95) 0.92	ab	86.76	a	104.4	ab
   S16	(0.13) 1.14	ab	84.50	a	97.9	a
   Ukulinga	(1.004) 1.25	abc	89.49	a	176.3	d
   SCI	(1.08) 1.27	abc	89.10	a	153.2	bed
   CI1	(1.06) 1.30	abc	96.82	a	147.3	abed
   S29	(1.10) 1.32	abc	92.83	a	165.2	cd
   004011	(1.20) 1.52	abc	91.32	a	122.7	abc
   RoyesB	(1.29) 1.78	abc	86.61	a	130.8	abed
   royesll	(1.39) 1.97	abc	88.44	a	127.6	abed
   WB74	(1.45) 2.17	be	96.19	a	102.1	ab
   G633I1	(1.49) 2.53	c	85.05	a	131.5	abed
   CV%	(27.9) 59.4		10.6		27.5
   LSD	(0.4704) 1.292		13.95		52.86
   SED	(0.2289) 0.628		6.76		25.88
   P	(0.165) 0.245		0.703		0.096

   • Results in parenthesis are transformed by square root transformation
   • Pink nodules are actively fixing nitrogen

   Table 2: Establishment of total rhizobium population in two different field sites

   	Control	Rhizobium	SED	CV%	P
   Ukulinga	83.9	96.2	4.08	5.6	0.096
   Baynesfield	95.82	9187	1.481	1.9	0.318

   • The total Rhizobium population was higher in the treated plants than in the control plants at Ukulinga.
   • But at Baynesfield, the untreated control showed higher total number of rhizobium than the treated one.

   Lab activity

   • Trichoderma harzianum was evaluated for its control efficacy against Aspergillus flavus in vitro.
   • In dual culture in vitro bioassay in Petri dishes showed a 61.5% growth inhibition of A. flavus by T. harzianum on potato dextrose agar (PDA).

   

   • Examination under Scanning Electron Microscope (SEM).
   • A scanning electron microscopical investigation of fungal interactions demonstrated no clear hyphae penetration of A. Flavus by Trichodenna spp.
   • This result suggest that mycoparasitism may not be the mechanism involved in the inhibitory interaction of A. Flavus with T. harzianum.

   

   Activity of the next two months

   • Shelling will be finished by the end of June.
   • Weighting and grainding (using coffee grainder) will be done in the first week of July.
   • Aflatoxin content will be quantified uisng ELASA by the end of July.
   • The paper will be ready for publication at the end of August.

2. Alternaria blight of sunflower and its control

   Prof TAS Aveling
   University of Pretoria

   Introduction

   The sunflower is a member of the Asteraceae, a large family of flowering plants occurring throughout the world, although a few are of economic importance. The sunflowers of the genus Helianthus have 67 species all of which are native to North and South America and 17 of which are cultivated (Weiss, 1983). Two of these species, H. annuus L., the common sunflower, and H. tuberosus L., the Jerusalem artichoke, are cultivated as food plants and several species are grown as ornamentals (Carter, 1978). In South Africa, the sunflower crop is the third most important field crop after maize (Zea mays L.) and wheat (Triticum aestivum L.) and is the most important oil crop (Grains South Africa, 2010). Other important oilcrops include the soybean [Glycine max (L.) Merr.], rapeseed (Brasicca rapa L.), and peanut (Arachis hypogaea L.) (Carter, 1978).

   Alternaria species are ubiquitous and are highly resistant to adverse weather, because they can develop under a wide range of temperatures and can utilize locally available sources of moisture (Green and Bailey, 2000). The genus Alternaria includes nearly 300 species that occur worldwide (Rotem, 1994; Pryor and Gilbertson, 2000) as either plant pathogenic or saprophytic species (Konstantinova et al., 2002). Alternaria leaf spot on sunflower (Helianthus annuus L.) is an important disease that can cause a significant reduction in crop yield (Allen et al., 1982). Alternaria helianthi (Hansf.) Tubaki and Nishihara is regarded as the main cause of Alternaria leaf spot of sunflowers. (Allen et al., 1983). Nine other Alternaria species have been reported on sunflower, including A. alternata (Fries) Kiessler, A. zinnae Ellis, A. tenuissima (Fries) Wiltshire, A. leucanthemi Nelen (syn A. chrysanthemi Simmons and Grosier), A. helianthicola Rao and Rajagopalan, A. longissima Deighton and MacGarvey, A. helianthinficiens Simmons, and A. protenta (Lapagodi and Thanassoulopoulos, 1998).

   The characterization and taxonomy of Alternaria species has been based on morphology and sometimes host association (Simmons, 1990; Rotem, 1994). Alternaria are divided into three major sections or groups according to their catenation i.e. Longicatenatae, in which conidia appear in long chains of about 10 spores or more; Brevicatenatae which forms short chains, usually of three to five spores; and Noncatenatae which form solitary spores that may be beakless, but may comprise species that have long beaks (Rotem, 1994; Chuo and Wu, 2002). When the genus as a whole is considered, species identification becomes more difficult because some species have ranges of spore dimension that overlap those of other species (Pryor et al., 2003). Many Alternaria species produce small spores aggregating in branching chains and collectively these species can be found in nearly all agricultural systems causing a variety of diseases (Agrios, 2005). Although the morphological differences in Alternaria species are subtle, the variations in pathogenicity may be great (Pryor et al., 2003). Catenation and conidial morphology of Alternaria species is affected by the conditions of growth such as substrate, light and humidity and thus may be unreliable both for implying phylogenetic relationships and characterization of various species (Rotem, 1994).

   Previously, Kusaba and Tsuge (1995) suggested that small-spored Alternaria should not be separated at species level but should be classified as Alternaria alternata with differentiation at species level as formae specialis. Recent studies by Lawrence et al. (2013) showed that molecular methods have been employed to classify or segregate Alternaria species, but with variable results. The molecular tools that have been used for Alternaria spp. include sequence analysis of GADPH and the Alt 1 gene. However, small-spored Alternaria species are still taxonomically a challenging group of fungi with few morphological or molecular characteristics that allow distinctive discrimination among the taxa (Andrew et al., 2009).

   Epidemics of Alternaria blight of sunflowers are most common and severe in areas that experience extended periods of wet weather in summer accompanied by mean daily temperatures between 25 and 30ºC (Reis et al., 2006). Alternaria species are reported to reduce seed and oil yield by 27 to 80% and 17 to 33%, respectively, and can cause germination losses (Calvert et al., 2005). The disease significantly reduces head diameters and numbers of seeds produced per head. The reduction in seed content caused by Alternaria blight is an economic concern, because growers receive a price premium or a dockage based on oil content (Carson, 1985).

   Aims and objectives

   • To determine the distribution of Alternaria blight by surveying the major sunflower growing areas in South Africa for Alternaria leaf spot.
   • To identify the causal agents of Alternaria blight based on morphology, toxin profiling and molecular techniques.
   • To determine the seed health and seed vigor of sunflower seed lots.
   • To determine location of infection by using a seed component test and Real-time PCR.
   • To determine the source of Alternaria species inoculums in sunflower farms.
   • To determine the susceptibility of different cultivars to Alternaria blight.
   • To determine bio-control and chemical control measures.
   • Microscopy (EM) study of infection process and bio-control and Alternaria interaction.

   Materials and methods

   SURVEY: THE DISTRIBUTION OF ALTERNARIA BLIGHT IN SOUTH AFRICA

   Surveys were conducted during 2012/13, 2013/14 and 2014/15 growing seasons on 60 sunflower production sites within all three seasons. The survey was conducted on cultivar trials managed by the ARC, seed companies and sunflower producer's farms. Four cultivars were selected for the cultivar trial survey (SY4045, PHB65A25, SY4200 and PAN7049) and 30 plants were randomly selected and surveyed on the producer's field. The plants were surveyed between 90 to 120 days after planting. Samples were collected for subsequent studies.

   Morphological and molecular identification

   Thirty Alternaria isolates were used in the present study that were obtained from leaf blight infected sunflower leaves and seed samples collected from major sunflower production areas of South Africa; North West, Limpopo, Free State, Mpumalanga and Gauteng provinces. The identities of the isolates were confirmed both by morphological and molecular techniques. The morphological identification of the isolates was observed and examined under Zeis stereomicroscope. The Alternaria spp. isolated from the various seed lots was compared to the three-dimensional sporulation pattern of the Alternaria spp. described by Simmons (1990; 1993; 1994; 1995). The thirty isolates were cultured on PCA for 7 days at 25ºC under 12hours alternating cycles of UV light and darkness.

   For molecular identification of the isolates, the genomic DNA was recovered using a CTAB-method (Ausubel et al., 1998). A 25 pi reaction volume containing a reaction mixture of 18.25 pi of sterile double-deionised water, 5U My Taq buffer, 0.25U Taq DNA polymerase, 0.25 pi of the respective primer sets (200 nM) and 1 pi template DNA (15 ng/pl) was used. Phylogenetic analyses were performed using PAUP v 4b 10. Gaps were treated as missing data. Analyses were conducted by heuristic searches consisting of 100 random addition replicates with branch swapping by tree-bisection-reconnection algorithm. Branch stability for individual dataset and concatenated dataset were evaluated by 1000 bootstrap replications using a heuristic search with simple sequence addition to produce a majority rule consensus tree with nodal support values. Congruency among datasets (P>0.050) was evaluated with the partition homogeneity test (PHT) implemented in PAUP v4.0. Alternaria helianthificiens was rooted as an outgroup in all the analyses based on results of Woudenberg (2013). A. helinathificiens was the most basal Alternaria group in the study. For each set of DNA sequences, phylogenetic trees will be constructed using distance methods.

   Pathogenicity tests

   Sunflower (Helianthus annuus L.) seeds (cultivar PAN 7351) were received from Pannar (PTY, LTD), Bapsfontein, South Africa. The plants were left to grow until 6-weeks-old before use in the subsequent experiments. Sunflower plants were then inoculated with a 4x10³ spores/ml conidial spore suspension of 2-weeks-old 30 Alternaria isolates. The evaluation of the infection was done over a period of 7 days. A disease severity index was compiled using symptoms from the pathogenicity test see Figure 3. Separation of means was done using the least significant difference (LSD) test (P<0.05). The disease severity was analysed using the standard analysis of variance (ANOVA) using SAS v9.4 (Institute, Inc., 2013) statistical package. All the representative cultures were submitted to the Agricultural Research Council – Plan Protection Research Institute, Mycology Unit (ARC-PPRI) for any further studies.

   Seed tests (standard germination, agar plate method, component test and seed treatment)

   Standard germination, seed health and seed component tests were done on 19 commercial sunflower seed lots received from various sunflower production areas of South Africa. The standard germination test will be conducted in rolled paper towels at 25ºC according to the rules of the International Seed Testing Association (2013). The first and second count of germination will be made after four and 10 days. Germination tests will be done as four replicates of 100 seeds. Alternaria species will be isolated from lesions of germinated sunflower seeds subsequent to the standard germination tests. Two hundred seeds of each of the 19 sunflower seed lots will be surface-disinfected with 1% sodium hypochlorite for 5 min, rinsed with sterile distilled water and placed on sterile paper towel in the laminar flow until dry. The seeds will be placed onto potato dextrose agar medium (PDA) (Merck) or sterilized blotter in Petri dish (used for blotter method), with five seeds in each Petri dish. The plated seeds will be incubated for 5-7 days at 25ºC under 12 hours alternating cycles of ultraviolet (UV) light and darkness.

   For seed component testing, each seed sample will be soaked in sterile distilled water for 7 to 8 hours at 25ºC. After the seed coat had softened, the seeds will be cut open to separate the cotyledon / embryo from the seed coat. The seed coat and cotyledon / embryo of the various sunflower cultivars will be plated out. Furthermore, to determine a possible fungicide for treating sunflower seeds, two seed cultivars were treatedwith six fungicides; Celest, Dynasty, Redigo, Apron XL, Dividend, Galmano and a bio-control agent IntegralP (Bacillus amyloliquefaciens MBI600) at both the recommended rate and twice the recommended rate.

   Field trials

   Field trials that were done focused on two aspects of disease control which include: (i) seed treatment; and (ii) plant spraying programs. Two field trials have been drafted and done in December 2014 (early planting date) and January 2015 (late planting date). Table 1 and 2 below will gives a general idea of the layout of the field trial seed treatment and spray program respectively. For seed treatment sunflower seeds were treated with five fungicides; Celest, Dynasty, ApronXL, Dividend, Galmano and a bio-control agent IntegralP (Bacillus amyloliquefaciens MBI600) at both the recommended rate and double the recommended rate. For spray program three spray fungicides were used as treatment: Duett, Abacus and AmistarTop. Six evaluation and ratings were done every two weeks within the growing season of the each field trial. The respective trials were harvested at the end of April and early July. Evaluation of the efficacy of the fungicide was based on disease severity in the field, Alternaria infection of progeny seeds, seed weight and sunflower head size. Statistical analysis will be done to conclude the evaluation the three field trials. One more trial will be done in December 2015.

   Results and discussion

   SURVEY: THE DISTRIBUTION OF ALTERNARIA BLIGHT IN SOUTH AFRICA

   The 2012/13 national cultivar trial results showed that Alternaria leaf blight disease severity on sunflower ranged between 44-54% in Bainsvlei, 50-61% in Delmas, and 51-81% in Potchefstroom in four plantings. Other surveyed localities ranged from 44-81%. The results of the severity of Alternaria leaf spot ranged from 18-60% in all sunflower producer's fields. However, in the 2013/14 season survey a slight increase in disease severity of the national cultivar trials was observed which ranged between 8-33% in Wesselbron, 43-48% in Delmas and 16-86% in Potchefstroom, while farmer fields surveyed ranged from 8-86%. Other diseases that were present during the surveys were head rot, white rust and Sclerotinia which were present in different sunflower production areas whereas diseases such as brown rust and stalk rot were each observed at specific localities.

   The 2013/2014 showed an increase in Alternaria blight severity both in cultivar trials and on production farms. This may be due to increased inoculum levels / sources, increased temperatures and rainfall experienced during the season or increased susceptibility. This survey demonstrated that Alternaria species are widespread across sunflower production areas in South Africa. The 2014/15 season results will be amended to the results once statistical analyses is done. To conclude this section of the study, statistical correlation between Alternaria disease severity and climatic conditions will be done. This chapter will help us better understand if the climate has a greater influence on disease severity. Thus, earlier/later planting dates may be recommended to sunflower producers.

   Identification and pathogenicity of Alternaria isolates

   Alternaria helianthi is generally regarded as the main cause of sunflower blight, but recently small-spored Alternaria have been found to cause severe leaf spots and blights. Pathogen and disease diagnosis are fundamental to virtually all aspects that relate to plant pathology (Ma and Michailides, 2007). Small-spored Alternaria species are increasingly becoming of great economic importance in the agricultural industry due to their cosmopolitan nature and ability to cause disease on non-host-crops (Pryor and Michailides, 2000). In this study, identification of these species was done using the morphological characterization according to the characteristics of conidia and sporulation patterns and molecular techniques using five gene regions that include ITS, GAPDH, RPB2 gene, Alt al and the TEF-la in order to support the morphological.

   Thirty Alternaria isolates were recovered from infected sunflower seeds and leaves collected during the surveys and studied under a stereo microscope. The Alternaria colonies growth pattern was circular with alternating circles due to the alternating 12 hours light and darkness regime and woolly texture (Fig 1). Conidiophore rose singly and ranged from 12 to 50 pm long, conidial width ranged from 8 to 15 pm and the length ranged from 12 to 34 pm. Based on the conidiophore size, conidia shape, conidia sizes three dimensional sporulation patterns the isolates were dived into three groups of A. tenuissima, A. alternata and A. helianthicola. There was no evidence of A. helianthi from all sampled seeds and leaves.

   • Culture and sporulation structures of Alternaria species on PCA isolated from lesions of sunflower (Helianthus annuus L.) infected seeds and leaves.
   • Sporulation pattern of Alternaria tenuissima (PCA 7d).
   • Sporulation pattern of Alternaria alternata (PCA 7d).
   • Sporulation pattern of Alternaria alternata (PCA 7d).
   • Sporulation pattern of Alternaria helianthicola (PCA 7d).

   

   Based on the phylogenetic analyses, the Alternaria isolates were separated into three types groups, A. alternata, A. tenuissima and an unknown Alternaria species, which was identified morphologically as Alternaria helianthicola. Koch's Postulates proved that the isolates could cause Alternaria leaf blight of sunflower as seen in the field. Alternaria helinathi was not isolated from any of the sunflower lesions.

   

   Following morphological and molecular identification, pathogenicity of the 30 isolates was tested in the greenhouse. A disease severity index was compiled to determine the disease severity of the Alternaria isolates during pathogenicity test. The pathogenicity test is presented in Figure 3. There was a significant difference in pathogenicity between the Alternaria isolates. Two types of disease symptoms were expressed. The first type of symptoms was seen as a small to large lesion surrounded by a chlorotic halo. The lesions were either found on the leaf tip or centre of the leaf. The second type of symptoms was seen as very tiny lesions that were closely packed to each other. These lesions were also seen to have a yellow halo. Pathogenicity tests showed that all the isolates were capable of causing Alternaria leaf blight disease although there was a significant difference between the isolates. Typical Alternaria lesions with chlorotic halo symptoms were observed during the evaluations. There was no observed geographical or symptoms expression grouping between the isolates. This implies that general Alternaria species that may be regarded as saprophytic can later be capable of causing disease of agricultural crops due to its non-host specific toxins (Pryor and Michailides, 2000) and that Alternaria species are as capable of causing disease despite their ecological or host association.

   • An asymptomatic sunflower leaf
   • Often symptoms begin as circular lesions on the inner part of the leaves or on the leaf margins.
   • As the time elapsed, the disease lesions became enlarged in size, and
   • as more lesions were formed, they eventually coalesced to form larger irregular lesions ...
   • to cause premature shrivelling of the leaves. The lesions are always surrounded by a chlorotic halo as soon as they are formed.

   

   Seed test

   Standard germination, seed health and seed component tests were done on 19 commercial sunflower seedlots received from various sunflower production areas of South Africa to determine whether seed infection by Alternaria spp. had an effect on the seed lots. Germination percentages ranged from 58 to 94%. Germination was found to be influenced by the severity of seed infection, although the correlation was a fairly weak (56%). Germination tests showed that the cotyledons of some of the seedlings after the germination tests had seedling blight lesions. The lesions were mostly circular with a dark brown centre. No chlorotic halo encircling the lesions was evident.

   The main fungal species that were present on the sunflower seed lots using the agar plate method were Alternaria followed by a low occurrence of Stemphylium, Rhizopus and Trichoderma species. Seedinfection by various small-spored Alternaria spp. ranged from 16 to 98% based on the agar plate tests. Seed component plating tests showed that the Alternaria species were more prevalent in the embryo and cotyledon than on the seed coats. The high seed infection rate during the agar plate tests and the infected cotyledon during germination tests may be an indication that small-spored Alternaria spp. are seed transmissible and may be a source of inoculum during a planting season.

   There is currently no fungicide treatment registered for the control of Alternaria blight on sunflower. In an attempt to determine an interim seed treatment, sunflower seeds were treated with six fungicides; Celest XL, Dynasty, Redigo, Apron XL, Dividend, Galmano and a bio-control agent Integral (Bacillus amyloliquefaciens MBI600) at both the recommended rate and twice the recommended rate. Seed treatments showed that Celest XL and Dynasty showed to be the most effective fungicides in reducing level of Alternaria infection. At the recommended dose, Apron XL did not have any effect on the disease, whereas Celest XL reduced the disease by 75%, Dynasty by 50% and Integral? and the other fungicides reduced infection by approximately 33%. Celest XL and Dynasty are known to be broad spectrum fungicides are registered to control Ascomycetes. Disease reduction did not differ significantly between the recommended rate and twice the recommended rate. These results will further be compared to the findings of the field experiments.

   Field studies evaluating the vigour of sunflower cultivars

   The preliminary observations noted in the field trial studies are that:

   • Disease starts being visible in the field from six weeks after planting.
   • There was a sporadic initial infection on the field which may be initiated by the high levels of inoculum in the seeds on that specific area of the field.
   • Of the two cultivars used in the field trial, the cultivar that was seen to have the most seed infection in the lab prior to planting generally had a more severe disease infection in the field and also during seed tests done post-harvest.
   • Temperatures and rainfall during the early planting date suits the proliferation of Alternaria and as result, the early planting date had greater Alternaria disease severity than the later planting date.
   • Spray treatment was more effective than seed treatment; unfortunately spray treatment may not be financially viable for farmers to use as a control for Alternaria in the field but spraying of sunflower crops can however be recommended for seed production companies to reduce level of seed infection.

   Subsequent to field trial, the seed yield has been taken to the lab for further evaluation. Yield weight, mass/1000 seed will be measured; seed health, seed germination and moisture content test are also currently being done as part of the evaluation. Results to lab evaluations will be given once statistical analysis of the all three trials is done.

   Table 1: Seed treatment trial plan

   Rep 1a
   C1F4D	C1F2	C2F5D	C2F0	C2F4	C2F1D	C1F3	C1F3D	C2F1
   Rep 1b
   C1B	C1F4D	C1F1	C1F1D	C2F3D	C2F2	C1F4	C1F1D	C1F0
   Rep 1c							Rep 2
   C2F2D	C1F5D	C2F3	C2F5	C1F5	C2B	Border	C1F2	C1F5
   Rep 2a
   C2F0	C1B	C1F0	C1F2D	C1F5D	C1F3	C2F2	C2B	C1F1
   Rep 2b
   C2F5	C1F1D	C1F3D	C2F5D	C1F4	C2F4	C2F3	C2F2D	C2F4D
   Rep 2c					Rep 3
   C2F1	C2F1D	C1F4D	C2F3D	Border	C2F2	C1F2	C1F3D	C2F1D
   Rep 3a
   C1F4	C1F2D	C2F3D	C1F1D	C1F3	C1F5D	C2F5	C2F4	C2F4D
   Rep 3b
   C1F1	C2F5D	C2F1	C2B	C1F0	C2F3	C1F4D	C1B	C2F0
   Rep 3c			Rep 4
   C2F2D	C1F5	Border	C1F1	C1F1D	C2B	C2F2D*	C2F3*	C1F5D
   Rep 4a
   C2F0	C2F4	C1F2D	C2F3D	C2F5	C1F2	C2F1	C1F3	C2F1D
   Rep 4b
   C1B	C1F4	C2F4D	C1F4D	C1F3D	C2F2	C1F0	C2F5D	C1F5
   Rep 5a
   C2F3	C1F2D	C1F5D	C2B	C1F1	C1F4	C1F3D	C1F3	C1B
   Rep 5b
   C1F2	C1F1D	C2F5	C2F4D	C2F1	C2F2D	C1F5	C2F0	C2F3
   Rep 5c							Rep 6
   C1F4D	C2F1D	C1F0	C2F2	C2F4D	C2F4	Border	C1F0	C2F2D
   Rep 6a
   C1F4D	C1F4	C1B	C2F5	C1F5	C2F3D	C2F2	C2F1	C2F4
   Rep 6b
   C1F1D	C1F1	C2F1D	C1F2D	C1F5D	C1F3	C2F5D	C1F2	C2F3
   Rep 6c
   C2F0	C2B	C1F3D	C2F4D	Border	Border	Border	Border	Border

   (3 rows per treatment, 6 reps, evaluation on middle row)

   Table 2: Seed treatment trial plan

   Rep 1
   C2S1	C2S2	C1S3	C2S3	C1S1	C1S0
   Rep 1			Rep 2
   C1S2	C2S0	Border	C2S0	C2S3	C1S1
   Rep 2
   C2S2	C1S2	C1S3	C1S0	C2S1	Border
   Rep 3
   C1S3	C2S1	C2S2	C1S0	C2S0	C2S3
   Rep 3			Rep 4
   C1S1	C1S2	Border	C1S1	C2S0	C1S2
   Rep 4
   C2S2	C2S3	C2S1	C1S0	C1S3	Border

   (5 rows per treatment 4 reps, evaluation on any of the 2 middle row)

   Abbreviations

   • Cl = Cultivar 1 (PAN 7057)
   • C2 = Cultivar 2 (PAN 7049)
   • F0 = control (Seed-treatment)
   • Fl = Dividend(Seed-treatment)
   • F2 = Galmano (Seed-treatment)
   • F3 = ApronXl (Seed-treatment)

   • F4 = Celeste (Seed-treatment)
   • F5 = Dynasty (Seed-treatment)
   • SI = Duett (Spray program)
   • S2 = Abacus (Spray program)
   • S3 = AmistarTop (Spray program)
   • B = Biocontrol (bacillus)

3. The role of seedling diseases in poor establishment of sunflower in South Africa

   Dr SC Lamprecht
   ARC-PPRI

   Poor establishment has been identified as one of the important constraints in sunflower production in South Africa. Although the contribution of other factors such as seedling vigour, seedbed preparation and soil temperature to poor establishment have been investigated, there is no information on the role of seedling diseases as a production constraint in sunflower production in South Africa. The main aim of this study is to determine the incidence of seedling diseases of sunflower and the major causal organisms associated with these diseases. The first phase of the project involves surveys and sampling of diseased sunflower seedlings and characterization of fungi associated with these seedlings. The current report includes results on surveys and sampling of diseased sunflower seedlings and fungi associated with cotyledons, hypocotyls and roots of these seedlings during December 2015 and February and March 2016 in the Free State, Limpopo, Mpumalanga and North West. Field trials were established with treated (Celest XL + Cruiser Maxx) and untreated seed of cultivar PAN 7102 CL at three localities each in the Free State and North West, and one each in Limpopo and Mpumalanga. Farmer fields planted to only treated seed were planted were sampled at three localities each in the Free State, and four each in Limpopo and North West province. Seedlings (60 per locality) and rhizosphere soil were sampled within six weeks after planting. Cotyledon, hypocotyl and root rot severities were recorded. There was often a clear difference in survival of seedlings in plots planted to treated and untreated seeds, with more seedlings that survived in plots planted to treated compared to untreated seed. It was also found that in certain instances seed did not germinate or showed poor germination. Similar to the previous year, stunted seedlings were present in all fields sampled. Symptoms on seedlings included lesions oncotyledon, hypocotyls and roots. At one locality in the Free State, root knot nematode was also recorded on seedling roots. In field trials, cotyledon and root rot was more severe in samples collected from the Free State and North West and at one of the localities in the Free State (Krst – Kroonstad) seed treatment significantly reduced the severity of cotyledon rot. Seed treatment only significantly reduced hypocotyl rot severity at the 2 Potch locality in North West province, but had no effect on root rot severity in any of the localities. Analyzing the samples obtained from farmer fields and field trials planted only to treated Poor establishment has been identified as one of the important constraints in sunflower production in South Africa. Although the contribution of other factors such as seedling vigour, seedbed preparation and soil temperature to poor establishment have been investigated, there is no information on the role of seedling diseases as a production constraint in sunflower production in South Africa. The main aim of this study is to determine the incidence of seedling diseases of sunflower and the major causal organisms associated with these diseases. The first phase of the project involves surveys and sampling of diseased sunflower seedlings and characterization of fungi associated with these seedlings. The current report includes results on surveys and sampling of diseased sunflower seedlings and fungi associated with cotyledons, hypocotyls and roots of these seedlings during December 2015 and February and March 2016 in the Free State, Limpopo, Mpumalanga and North West. Field trials were established with treated (Celest XL + Cruiser Maxx) and untreated seed of cultivar PAN 7102 CL at three localities each in the Free State and North West, and one each in Limpopo and Mpumalanga. Farmer fields planted to only treated seed were planted were sampled at three localities each in the Free State, and four each in Limpopo and North West province. Seedlings (60 per locality) and rhizosphere soil were sampled within six weeks after planting. Cotyledon, hypocotyl and root rot severities were recorded. There was often a clear difference in survival of seedlings in plots planted to treated and untreated seeds, with more seedlings that survived in plots planted to treated compared to untreated seed. It was also found that in certain instances seed did not germinate or showed poor germination. Similar to the previous year, stunted seedlings were present in all fields sampled. Symptoms on seedlings included lesions on cotyledon, hypocotyls and roots. At one locality in the Free State, root knot nematode was also recorded on seedling roots. In field trials, cotyledon and root rot was more severe in samples collected from the Free State and North West and at one of the localities in the Free State (Krst – Kroonstad) seed treatment significantly reduced the severity of cotyledon rot. Seed treatment only significantly reduced hypocotyl rot severity at the Potch locality in North West province, but had no effect on root rot severity in any of the localities. Analyzing the samples obtained from farmer fields and field trials planted only to treated seedlings collected from localities in Limpopo and North West, and Rhizopus spp. More frequently from seedlings collected in the Free State and Mpumalanga. There were no significant differences in the incidences of F. equiseti and Trichodermci spp. on seedlings collected from the four provinces. Soil samples collected from the different localities were split in half and one half was pasteurized to eliminate soilbome pathogens. Both pasteurized and non-pasteurized soils were planted to treated (Celest XL + Cruiser Maxx) and non-treated seed of cultivar PAN 7102 CL under glasshouse conditions. Seedling survival, seedling length, cotyledon, hypocotyl and root rot severity were recorded. Many interactions were recorded for seed treatment, soil pasteurization localities and provinces, but in general seed treatment improved survival of seedlings and reduced growth of seedlings, although not in soil from all localities. Seed treatment only significantly reduced root rot severity of seedlings in soils collected in the Free State. Unfortunately seed treatment caused premature dying of cotyledons of seedlings in the glasshouse. Soil pasteurization significantly reduced cotyledon and root rot severity in the soils from all provinces and significantly increased growth of seedlings in soil from Mpumalanga. Plant length was improved by soil pasteurization although not always significantly and seed treatment only significantly improved plant length in soil collected from Mpumalanga. The results of this survey showed that cotyledon, hypocotyl and root rot occur in young sunflower seedlings in the major sunflower production areas and that pathogens were obtained from seedlings with disease symptoms that can significantly affect seedling health. Furthermore, certain pathogens were more prevalent on certain plant parts and also more prevalent in certain localities or provinces. Some of the fungi isolated appears to be new records on sunflower. The preliminary pathogenicity test identified species within Fusarium, Pythium and Rhizoctonia that can be responsible for poor establishment of sunflower seedlings, but these results need to be confirmed and should include representative isolates of all potential pathogenic fungi isolated during the 2014/15 and 2015/16 surveys. Also soil pasteurization to eliminate soilbome pathogens significantly reduced cotyledon and root rot, but seed treatment with Celest XL + Cruiser Maxx was less effective in reducing disease symptoms. The results clearly demonstrate the complexity of pathogens associated with sunflower seedlings and that different complexes are present in different production areas. Once the most important pathogens of sunflower seedlings are identified it will be important to evaluate the efficacy of the Celest XL + Cruiser Maxx treatment against these pathogens and whether it is necessary to improve the seed treatment to target seedling disease complexes to significantly improve seedling health and establishment of sunflower in South Africa.

4. Funding of the Supply and Demand Estimates Committee

   Mr C Joubert
   NAMC

   The primary objective of this report is to provide stakeholders in the industry with a balanced and holistic view of the activities and impact of the Supply & Demand Estimates Committee (S&DEC). The document also reports on expenditure and income of the Supply and Demand Estimates initiative for the 2015/2016 season. But first, the S&DEC want to sincerely thank all stakeholders who contributed to the Supply and Demand Estimates initiative. It is important to note that without the funding, contribution of the co-workers and the committee members it would have not be possible to provide the necessary information as to date.

   The total cost for the initiative for the period from 1 Apr 2015 to 31 March 2016 amounted to R515 958, against a budget oft R795 000. The Grain & Oilseeds Supply & Demand Estimate initiative was financed by the NAMC for this period.

   A South African Supply & Demand Estimates (SASDE) report was published every month, ina specific format prescribed by the S&DELC. The reports provide detailed informationregarding the beginning stocks, supply (production, retentions and imports), demand (consumption and exports) and ending stock levels. These reports were widely accepted andused by grain, oilseeds and other stakeholders in the industry and government.

   Participation regarding the collection of applicable data can improve. It was also for this reason that the S&DEC recommended the implementation of a statutory measure to the Grain & Oilseeds Supply & Demand Estimates Liaison Committee (S&DELC). See Appendix 1 for letter of recommendation. This was discussed in detail at a S&DELC meeting on 11 February 2016. It was decided to keep to the "status quo" for now.

   The NAMC appointed a grain specialist via an appointment committee selected by the S&DELC and the NAMC. Dr Abongile Balarane started on 1 Jun 2015 in this position.

5. Website

   Ms M du Preez and Ms Y Papadimitropoulos
   OAC/OPDT

   The staff and webmaster, Tigme.com, paid significant attention to our web page to improve its user-friendly approach and further improve its efficacy as communication tool.

   Information available on the web page include the guidelines and application forms for research projects and bursaries, information about achievement awards, crop estimates and minutes of forum meetings.

   The publication of research results on the web page enjoyed continuous attention during the year.

   The OAC/OPDT is satisfied with the progress and the utility value of the web page.

6. Oilseeds information

   Mr N Hawkins
   SAGIS

   Forums and Trusts

   The General Manager, Mr Nico Hawkins, attended the PRF Soybean Work Group, Groundnut Forums, Sunflower and Soybean Forums where SAGIS' information was presented and distributed to all role players.

   SAGIS' Board of Directors

   The Oil and Protein Seeds Development Trust (OPDT) and Oilseeds Industry were represented on SAGIS' Board of Directors, by Mr GJH Scholtemeijer and Mr JDW Boshoff with Ms JM van der Merwe as their alternate.

   Dr JL Purchase and Mr AAA Nebe were the Chairperson and Vice Chairperson.

   Product Information

   The publication dates for product information are available on SAGIS' website. Statutory measures for oilseed products were approved.

   Information

   For further information on SAGIS' publication dates, information published, annual report, presentations, etc. refer to our website.

7. Oilseeds South African soybean crop quality survey

   Ms W Louw
   SAGL

   The 2014/2015 production season was extremely trying for producers with wet and dry periods alternating outside the normal patterns. During the harvesting season, a representative sample of each delivery of soybeans at the various silos was taken according to the prescribed grading regulations. One hundred and fifty composite soybean samples, proportionally representing the different production regions, were analysed for different quality parameters. The samples were graded, milled and chemically analysed for moisture, protein, fat and ash content. Fifteen randomly selected samples were analysed for genetic modification.

   The goal of this crop quality survey is to accumulate quality data on the commercial soybean crop on a national level. This valuable data reveal general tendencies, highlight quality differences in commercial soybeans produced in different local production regions and provide important information on the quality of commercial soybeans intended for export. With this data, SAGL is building up a database with quality data over different production seasons which can be used for decision making processes. This is the fourth annual soybean crop quality survey performed by The Southern African Grain Laboratory NPC. The results are available on the SAGL website. The hard copy reports are distributed to all the Directly Affected Groups and interested parties. The report is also available for download in a PDF format from the website. The 2014/2015 Report of the National Soybean Cultivar trials conducted by the ARC-Grain Crops Institute is also included in the report, as is the national grading regulations as published in the Government Gazette of 20 June 2014.

   Summary of results

   Eighty-seven percent (131) of the 150 samples analysed for the purpose of this survey were graded as Grade SB1 and 19 of the samples were downgraded to COSB (Class Other Soya Beans). Based on the samples received for this crop survey, Sclerotinia did not pose any problems. The highest percentage of Sclerotinia observed (0.2%) was on a sample from Mpumalanga, which is well below the maximum permissible level of 4%. During this season, the samples from the Northern Cape had the highest weighted average percentage Sclerotinia (0.07%). The national weighted average percentage this season was 0.01% compared to the 0.03% of the previous three seasons.

   North West province (18 samples) reported the highest weighted average percentage soybeans and parts of soybeans above the 1.8mm slotted sieve which pass through the 4.75mm round hole sieve, namely 2.15%. Limpopo (2 samples) reported the lowest at 0.67%. Mpumalanga province with the highest number of samples (77) reported an average of 1.78%. The Free State province averaged 1.77% (42 samples).

   The nutritional component analyses, namely crude protein, -fat, -fibre and ash are reported as % (g/100g) on a dry/moisture free basis (db). The weighted average crude protein content this season was 39.89%, comparing very well with the 39.84% of the 2013/2014 season. Mpumalanga showed the highest weighted average crude protein content of 40.44%. The weighted average crude fat content decreased from 19.78% in 2013/2014, to 19.3% this season. The samples from Limpopo had the highest weighted average crude fat content of 23.6%. The lowest average fat content was observed in Gauteng with 18.9%. The ash content did not vary significantly over the last four seasons, 4.64% this season compared to 4.66% 4.65% and 4.62% for the previous three seasons.

   All fifteen samples analysed, tested positive for the presence of the CP4 EPSPS trait (Roundup Ready®).

   

8. Oilseeds South African sunflower crop quality survey

   Ms W Louw
   SAGL

   This was the third annual national sunflower crop quality survey performed by The Southern African Grain Laboratory NPC.

   During the harvesting season, a representative sample of each delivery of sunflower seeds at the various silos was taken according to the prescribed grading regulations. The sampling procedure as well as a copy of the grading regulations form part of the report. One hundred and seventy six composite sunflower samples, representing the different production regions, were analysed for quality. The samples were graded, milled and chemically analysed for moisture, crude protein, crude fat, crude fibre as well as ash content.

   The goal of this crop quality survey is the compilation of a detailed database, accumulating quality data collected over several seasons on the commercial national sunflower crop, which is essential in assisting with decision making processes. The results are available on the SAGL website. The hard copy reports are distributed to Directly Affected Groups and interested parties. The report is also available for download in a PDF format from the website.

   Summary of results

   Eighty six percent (151) of the 176 samples analysed for the purpose of this survey were graded as Grade FH1 and twenty five of the samples were downgraded to COSF (Class Other Sunflower Seed). The percentage of FH1 samples showed an increase compared to the 82% and 80% of the 2013/2014 and 2012/2013 seasons respectively.

   • Twenty of the samples were downgraded as a result of the percentage of either the screenings or the collective deviations or a combination of both exceeding the maximum permissible deviations of 4% and 6% respectively.
   • Two of the samples were downgraded as a result of a combination of the foreign matter and collective deviations exceeding the maximum permissible deviations of 4% and 6% respectively.
   • Of the remaining three samples, one was downgraded due to the percentage damaged sunflower seeds exceeding the 10% maximum permissible deviation, one as a result of the presence of poisonous seeds (Datura sp.) exceeding the maximum permissible number (1 per 1000g) and the last sample was downgraded as a result of the presence of stones, glass, metal coal or dung.

   As in the previous season, the highest weighted percentage foreign matter (2.18%) was reported for the samples from Gauteng (N=5). The Free State and North West provinces averaged 1.15% and 1.16% respectively. The lowest average percentage was found in Limpopo at 0.17%. The RSA average of 1.17% was the lowest of the last three seasons.

   Sclerotinia did not pose a problem on any of the samples received for this survey and was observed only on nine of the samples. The highest percentage (3.03%) was present on a sample from Gauteng, this is however still well below the maximum allowable level of 4%. The national average of 0.04% compared well with the 0.01% of the 2012/2013 season and was lower than the 0.53% of the previous season.

   Test weight does not form part of the grading regulations for sunflower seed in South Africa. An approximation of the test weight of South African sunflower seeds is provided in Table 1 for information purposes. The g/1 L filling weight of sunflower seed was determined by means of the Kern 222 apparatus. The test weight was extrapolated by means of the following formulas obtained from the Test Weight Conversion Chart for Sunflower Seed, Oil of the Canadian Grain Commission: y = 0.1936x + 2.2775 (138 to 182g/0.5 L) and y = 0.1943x + 2.1665 (183 to 227g/0.5 L).

   Table 1: Approximation of test weight per province over three seasons

   Province	Test weight, kg/hl
   	2014/2015 Season			2013/2014 Season			2012/2013 Season
   	WA	Range	NS	WA	Range	NS	WA	Range	NS
   Free State (Regions 21-28)	44,1	38.9-49.9	69	41,8	36.4-48.2	*96	43,8	38.3-47.7	58
   Mpumalanga (Regions 29-33)	41,9	35.0-42.2	8	37,6	35.0-42.2	5	42,5	38.1-45.7	6
   Limpopo (Region 35)	43,9	42.2-50.5	8	42,4	37.7-44.0	11	44,6	42.6-47.5	9
   Gauteng (Region 34)	44,8	42.2-47.6	5	42,8	41.7-44.6	4	42,7	42.6-42.8	2
   North West (Region 12-20)	44,5	34.0-48.9	86	40,2	31.1-46.6	58	43,0	31.5-47.3	77
   RSA	44,2	34.0-50.5	176	41,3	22.6-48.2	174	43,4	31.5-47.7	152

   Abbreviations:   WA – Weighted Average  |  NS – Number of Samples

   * Two samples with outlier values as a result of Deviations (Screenings + Sclerotinia + Foreign matter) exceeding 18%, was not taken into account for calculation purposes.

   The nutritional component analyses, namely crude protein, -fat, -fibre and ash are reported as % (g/100g) on an "as received" or "as is" basis. The weighted average crude protein content of the 2014/2015 season was 16.96%, 0.81% higher than the previous season and 0.17% higher than in the 2012/2013 season. North West had the highest weighted average crude protein content of 17.53% and the Free State the lowest with 16.27%. Mpumalanga's crude protein content averaged 16.47%. The weighted average crude fat percentage of 39.7% compared very well with the 39.6% and 39.2% of the two previous seasons. Gauteng had the highest weighted average crude fat content of 41.4%. The lowest average fat content was observed in Limpopo (38.8%). North West and the Free State averaged 39.2% and 40.4% respectively.

   The weighted average percentage crude fibre decreased slightly from 20.2% in the previous season to 20.0% this season and equalled the 2012/2013 value. The values varied between 19.1% in Gauteng to 20.7% in Mpumalanga. The weighted average ash content is slightly lower (2.55%) than last season (2.66%) but similar to 2012/2013 (2.54%). The provincial averages ranged from 2.45% in Gauteng and Mpumalanga to 2.58% in the Free State.

   Table 2 provides a comparison between the South African Crop Quality Averages between the 2014/2015 and 2013/2014 seasons:

   Table 2: South African sunflower crop quality averages 2014/2015 vs 2013/2014

   Class and Grade Sunflower	2014/2015			2013/2014
   	FH1	COSH	Average	FH1	COSH	Average
   Grading
   1. Damaged sunflower seed, %	0.17	0.93	0.27	0.38	4.24	1.06
   2. Screenings, %	1.53	5.20	2.05	1.26	3.71	1.69
   3. Sclerotinia, %	0.05	0.00	0.04	0.13	2.43	0.53
   4. Foreign Matter, %	1.08	1.70	1.17	0.90	3.58	1.38
   5. Deviations in 2, 3 and 4 collectively. Provided that such deviations are individually within the limits of said items, %	2.66	6.90	3.26	2.29	9.72	3.60
   Musty, sour, khaki bush or other undesired smell	No	No	No	No	No	No
   Substance present that renders the seed unsuitable for human or animal consumption or for processing into or utilization thereof as food or feed	No	No	No	No	No	No
   Noxious seeds (Crotalaria sp., Datura sp., Ricinis communis)	0	0	0	0	0	0
   Noxious seeds (Argemone mexicana L., Convolvulus sp., Ipomoea purpurea Roth., Lolium temulentum, Xanthium sp.)	0	0	0	0	0	0
   Number of samples	151	25	176	145	31	176
   Chemical Analysis
   Moisture, % (5hr, 105 °C)	4.7	4.5	4.7	2.9	3.2	3.2
   Crude Protein, % (as is)	17.06	16.34	16.95	16.19	15.99	16.15
   Crude Fat, % (as is)	39.5	41.1	39.7	39.8	38.7	39.6
   Ash, % (as is)	2.56	2.52	2.55	2.65	2.70	2.66
   Crude Fibre, % (as is)	20.0	19.6	20.0	20.0	20.8	20.2
   Number of samples	151	25	176	145	31	176

   

9. Enhancing the production potential of groundnuts in South Africa by evaluating im­ported high yielding cultivars

   Ms L Salomon
   ARC-GCI

   The aim of the project was to evaluate new introductions for yield and seed quality under dryland and irrigation conditions. Advanced breeding lines from the ARC groundnut breedingprogramme were also included. Although Phase 1 Trials are in most cases a continuousprocess, a period of three seasons was completed to evaluate the stability of cultivars and lines and their reliability for yield. Due to severe drought conditions a total of four trials wereused to compile the data for this report. All trials consisted of 26 cultivars and lines. Once yield and grading quality were taken into consideration, Int 014, PC 435-K5, and Int 002 performed the best over all the localities and treatments. Taking yield stability into consideration over a three-year period over all treatments as well as locations PC 435-K5 (ARC-SelliePlus) and PC 478-K5 (ARC-AkwaPlus) were the top performers. South Africa must strive to produce a high quality groundnut that will not only ensure marketability of the product in international markets but also profitability for the producer at ground level for the producer at ground level.

10. Response of sunflower to a conservation agriculture production system and ni­tro­gen fertilization

    Dr AA Nel
    ARC-GCI

    There is a worldwide shift from conventional tillage crop systems towards conservation systems where no-till is practiced. For crops such as maize, it is recommended that the nitrogen fertilisation rate should be higher in no-till than in conventional tilled systems. It is unknown how sunflower would respond to no-till locally and if it requires a higher nitrogen fertilisation rate. The objective of this project is to investigate these two aspects and determine if nutrient uptake, diseases, pests and weeds are different in the tilled and no-till systems. A field trial was established on a sand clay loam textured Avalon soil at Potchefstroom in November 2013. Treatments were till and no-till as main plots and four nitrogen fertilisation rates allotted to sub plots. Nutrient concentration in the biomass and total uptake were affected in most seasons during one or more growth stages by one or, by both treatment factors. This indicates that nutrient uptake is affected by a seasonal weather, especially rainfall, interaction with tillage and nitrogen fertilisation. No differences in diseases, pests and weeds were observed between tillage systems, nor among nitrogen fertilisation rates. No indication could be found that tilled and no-till sunflower crops have different nitrogen fertiliser requirements. Over the three consecutive seasons, the yield of the no-till sunflower improved from 15% below to 34% above the yield if the tilled system.