GrainGenes
A Database for Triticeae and Avena
H.A. Smit, B.L. de Villiers, C.F. Pool, and R.C. Lindeque.
Water quality and adjuvant studies.
A combination of methylated sunflower oil (0.5 %) and ammonium
nitrate (1 %) was the most effective adjuvant combination with
tralkoxydim. Ammonium nitrate probably overcame antagonism from
sodium bicarbonate, and the methylated sunflower oil could have
increased the rate of tralkoxydim absorption.
Glyphosate was evaluated in water carriers containing
high levels of calcium chloride. Ammonium sulphate proved to
be just as efficient in overcoming the antagonism as a local acidifier
(Bladbuff 5 or Indicate 5). Both cationic and nonionic surfactants
were efficient with glyphosate. Methylated seed oils were efficient
only with glyphosate when applied in combination with ammonium
sulphate.
J.T. Steyn and J.P.C. Tolmay.
Field trials were conducted in the Central and Eastern
Free State to determine the effect of soil tillage on soil water
storage, soil compaction, root development, plant growth, and
yield. Four tillage treatments were used: conventional, two minimum
tillage systems, and chisel plow.
No differences in soil water storage or use could
be obtained with the four different tillage systems. Soil compaction
increased in resistance (kPa) with all four tillage treatments,
especially with conventional tillage where a plow pan formed.
Cultivar differences in root development were obtained, but no
tillage differences could be measured in root development.
Conventional tillage showed better plant growth
and development and a higher leaf area index over two seasons
in the Eastern Free State. In the Central Free State, no plant
growth differences occurred. Minimum tillage resulted in better
yields in the Central Free State whereas conventional tillage
yielded better in the Eastern Free State.
Publications.
Steyn JT, Tolmay JPC, Human JJ, and Kilian WH. 1995.
The effects of tillage systems on soil bulk density and penetrometer
resistance of a sandy clay loam soil. SA J Plant & Soil
12(2):86-90.
Steyn JT, Tolmay JPC, and Human JJ. 1995. The influence
of tillage on early root development and biomass of wheat cultivars
(Triticum aestivum L.). SA J Plant & Soil 12(4):147-151.
Development of eyespot-resistant wheat cultivars.
D.B. Scott, K. Wilkin, C. de Villiers, and H.J.L. Potgieter.
Isoelectric focusing gels were used to distinguish
between the seed of susceptible and resistant lines and also between
homozygous resistant and heterozygous resistant lines. Endosperm
from each seed to be tested for resistance was suspended in water
and spotted on a starch gel plate. Isozyme patterns of susceptible
and resistant lines were compared. More than 300 gels were prepared
to evaluate the progeny of the different backcrosses for resistance.
During 1994, backcrosses of the original F1 plants progressed
to the BC9, BC8, and BC4 of homozygous resistant lines of SST66,
Palmiet, and W84/7, respectively.
Fungicide trials. Spray
treatments of wheat with tebuconazole and carbendazim were repeated
at three localities in the Western Cape Province. Palmiet, planted
at Riviersonderend on 17 May, 1994, responded positively to both
fungicides when nitrogen fertilizers were applied in low amounts
(30 kg N/ha). Yield increases were obtained with two applications.
At Philadelphia, yields increased significantly with two applications
of the fungicides, but not with one application. Yield increases
occurred in the absence of fungal diseases on the leaves, but
physiological leaf spot was present at both localities. As a
result of a lack of early rain, trials at Malmesbury could not
be planted before the end of May. At this site, foliar diseases
did not develop, and yields were unaffected by fungicide sprays.
Biological control of root diseases.
During a survey of rhizobacteria in the soil, manganese-reducing
strains of Bacillus were commonly isolated from heat-treated
soils and from soils that are suppressive to take-all. About
200 isolates of manganese-reducing bacteria were isolated from
the rhizosphere of healthy wheat plants. Most isolates represent
Bacillus spp. Some isolates caused growth stimulation
of wheat plants in pots when unsterilized soil was inoculated
with a bacterial suspension and fertilized with both superphosphate
and limestone ammonium nitrate. With adequate nitrogen application,
most isolates used for seed treatment were able to promote growth
of wheat in a field trial near Bethlehem. A strong degree of
antibiosis to Ggt was displayed by one isolate of B. subtilis,
which shows that isolates of this species have great potential
for biocontrol of take-all in wheat.
Deleterious rhizobacteria.
Fluorescent pseudomonads were isolated commonly from soils that
are conducive to take-all of wheat. On agar media, these bacteria
were unable to reduce the manganese-oxidizing ability of Ggt.
They are considered as manganese-oxidizers, even though they
produce organic acids that reduce manganese around colonies on
a potassium permanganate medium. Results obtained in field trials
at Bethlehem showed that fluorescent pseudomonads are deleterious
rather than beneficial to wheat. Deleterious rhizobacteria were
favored by wet conditions and the presence of plant residue in
the soil. Root exudates of oat plants restrict development of
fluorescent pseudomonads, and apparently, this is the reason for
the good performance of wheat after oats.
UNIVERSITY OF THE ORANGE FREE STATEóDEPARTMENT OF BOTANY AND GENETICS
Faculty of Science, Bloemfontein, 9300, South Africa.
Alexander A. Myburg, Anna-Maria Botha, Brenda Wingfield*
*Department of Microbiology, University of the Orange
Free State.
A number of potential RAPD markers linked to RWA
resistance and to specific resistant and susceptible cultivars
were identified and are being characterized in this study. The
linkage of these molecular markers to resistance genes and their
potential use in the wheat breeding program are being investigated
currently. The aim of the study was to investigate the potential
use of these molecular markers to clone the RWA-resistance genes.
The first phase of the study involved the identification
of potential RAPD markers for RWA resistance. A total of 180
oligonucleotide primers (Operon Technologies primer kits A-I)
was screened in order to identify RAPD markers for the Dn1
resistance gene. Six susceptible wheat cultivars, six isogenic
resistant lines, and seven resistance source cultivars were screened
to study the inheritance of the RAPD markers obtained. A segregating,
F2 progeny population of the susceptible (Palmiet) and resistant
(SA1684) parents was analyzed to determine linkage to the resistance
gene. In the second phase of the project, specific RAPD fragments
are being cloned and characterized in order to construct sequence-characterized
regions (SCARs) that can be used as specific molecular markers
for RWA-resistance and as molecular tools for cloning the resistance
gene(s).
A total of 157 scorable RAPD loci were amplified
using 23 oligonucleotide primers. Of these, 30 % were present
in all of the cultivars, whereas 46.4 % displayed informative
polymorphisms. The majority (> 60 %) of the informative polymorphisms
distinguished the out-group (a rye cultivar). The low level of
polymorphism observed between the susceptible and resistant cultivars
in the study explains the problems experienced in generating closely
linked RAPD markers.
One combination of near isogenic lines (NILs), Palmiet
and Palmiet Dn1, was screened with 120 RAPD primers (Operon
Technology kits OPAñOPF). No repeatable polymorphisms
were generated by this approach, and bulk segregant analysis (BSA)
was used subsequently to analyze a segregating F2 population of
95 plants. Eight and 16 plant bulks were constructed, and the
resistant and susceptible bulks were screened with a total of
180 primers (OPAñOPI). Only two primers yielded repeatable
polymorphisms. One of these, OPC4-480, was a major polymorphism,
but was present only in the susceptible bulks. The other primer
generated a minor polymorphism, OPA17-500 that was present only
in the resistant bulks. Linkage analysis of the two markers yielded
a linkage distance of 27 cM for OPC4-480. The resistance-linked
marker OPA17-500 yielded ambiguous results when used to analyze
the 95 individual plants in the F2 population. The marker was
absent in all of the susceptible plants but present in only 30
% percent of the resistant plants. Both markers were used in
Southern analysis of wheat genomic DNA and displayed annealing
patterns typical of low copy number DNA (three and one copies,
respectively).
The two RAPD fragments were isolated from agarose
gels, reamplified, purified, and blunt-end ligated into the plasmid
vector pGEM3Z. The ligated plasmid mixture was transformed competent
cells of E.coli strain JM109. Positive clones were identified
by direct RAPD analysis of cell samples of the bacterial colonies.
Positive clones of the RAPD fragments are now being sequenced
in order to design and construct longer (more specific) primers
that can be used to amplify SCARs linked to the Dn1 resistance
gene.
The results generated by the study thus far indicate
that the RAPD technique can be used to generate molecular markers
for Russian wheat aphid resistance in wheat. Repeatability of
RAPD polymorphisms is still a major problem because of the large
number of parameters influencing the RAPD profile obtained from
each amplification reaction. We have successfully optimized and
standardized the RAPD conditions for generating repeatable profiles.
Unfortunately, more RAPD primers will have to be screened in
order to identify resistance markers more closely linked to the
resistance gene(s) (< 2 cM). At present, we are purchasing
additional primer sets and will use these to screen for RAPD markers
more closely linked to the Dn1 gene. In addition, we are
screening NILs and F2 bulks of the Dn2 and Dn5 RWA-resistance
genes for useful polymorphisms.
Effect of Russian wheat aphid on the expression of chitinase.
M.A.C. Nagel, A-M. Botha, and F.C. Botha*.
*Department of Biotechnology, University of Natal,
Mont Edgecombe, 4300, South Africa.
Plants respond to various pests and pathogens by
altered patterns of protein synthesis where enzymes, whose apparent
function is to defend the plant against all types of invaders
(pests and pathogens), are synthesized. One of these pathogenesis-related
(PR) proteins has been identified as chitinase (EC 3.2.1.14).
Chitinase activity was determined, in resistant and susceptible
T. aestivum, after Russian wheat aphid (Duiraphis noxia)
infestation, ethylene treatment, and mechanical wounding. Seven
days after infestation by aphids, a major induction in endochitinase
activity was measured in the apoplastic fluid of the resistant
plants. This induction in activity was not the direct result
of wounding, because exogenously applied ethylene (15 nl/l) induced
chitinase only in susceptible plants, whereas no substantial differences
were observed after mechanical wounding.
Western blot analysis revealed the existence of
four acidic apoplastic chitinases and four basic vacuolar chitinases
with molecular masses between 36.0, 34.0, 27.0, and 22.0 kD and
61.0, 54.0, 49.5, and 35.1 kD, respectively. Eight chitinase
isoforms were detected in the control plants, and three additional
isoenzymes were induced by the different treatments tested. The
pI values for these isoenzymes ranged from 3.5ñ9.5, indicating
that the different isoforms can be divided into acidic and basic
chitinases. After D. noxia infestation, two additional
peaks (pI 3.4 and 5.5) with chitinolytic activity were observed
in the resistant plants. Infestation also caused a massive accumulation
of a chitinase isoenzyme with a pI value of 4.1. The activity
of this chitinase isoform was fourfold higher in resistant plants.
This isoform also increased fivefold in the ethylene-treated
susceptible plants. Therefore, the expression patterns of the
different isoforms can be characterized into three groups: low
constitutive, newly induced, and enhanced constitutive. The results
of this study indicated that infestation by D. noxia, ethylene,
and mechanical wounding are separate, independent signals for
the induction of chitinase.