Methods for inhibiting rust infections of plants

Methods are provided for inhibition of leaf rust infections of plants, especially wheat, using inhibitors which disrupt sulfhydryl bonds of the enzymes used by the leaf rust germ tube to ingest and metabolize components of epicuticular waxes.

BACKGROUND OF THE INVENTION 
This invention is generally in the field of methods for treatment of fungal 
infections in plants, especially rust infections of wheat. 
Rusts are pathogenic parasitic fungi which infect wheat, barley, oats, 
beans, corn, sorghum, and other plants. Each rust is generally specific to 
its host and the location on the plant where infection occurs. Stem rust 
(Puccinia graminis f. sp. tritici) is a fungus which principally infects 
the leaf sheath of wheat plants. Leaf rust (Puccinia recondita f. sp. 
tritici) infects wheat plants through the stomates. Stripe rust (Puccinia 
striiformis) is similar to leaf rust but differs in that infections appear 
systemic due to colonization patterns on wheat leaves. 
As shown in FIGS. 2 and 3, scanning electron photomicrographs courtesy 
University of Minnesota SEM lab, rust spores germinate on the waxy surface 
of the plant, forming germ tubes which migrate laterally across the 
surface to the stoma where an appressorium is formed. A structure known as 
an infection peg grows downward through the stoma from the appressoria 
following chromosomal and protein changes within the appressoria. The peg 
forms a substomatal vesicle from which infection hyphae ramify inside of 
the leaf. Continued internal development leads to formation of 
subcuticular uredia which produce reinfecting urediniospores which are 
wind-disseminated after uredia rupture the epidermis. 
Rust is a good parasite in the sense that it does not kill its host, but 
reduces yield by stealing nutrients from its host. The plant can be 
simultaneously infected with other parasites including smuts and other 
fungi. Powdery mildew, (Erysiphe graminis, also germinates, forms a germ 
tube, and rapidly develops an appressorium from which the peg is capable 
of directly penetrating the cuticle of the leaf, in contrast to the rust 
fungi which must penetrate the leaf through the stoma. Powdery mildew 
possesses a cutinase enabling it to effect direct penetration. 
In 1986 and 1987, approximately 126,000,000 bushels of wheat in the United 
States were lost to three rusts, stem rust, leaf rust, and stripe rust, an 
economic loss of greater than $378 million dollars. Each year, major 
losses occur in some of the nation's wheat-providing states. 
Methods presently in use to combat rust infections include 1) use of rust 
resistant cultivars, 2) topical application of fungicides, and 3) cultural 
practices. Unfortunately, due to the relatively high rate of mutation of 
the rust organism, completely new cultivars of wheat are needed every 
seven years. Fungicides, while effective, are expensive and must be 
applied as a preventative, even if it is not certain that the plants will 
be infected. Many of the compounds previously in use have been withdrawn 
by the EPA. Compounds which are now utilized are more easily degraded and 
therefore less harmful to the environment but are more acutely toxic to 
humans. Thus, fungicide applications are even less desirable than before. 
The disadvantages and lack of success of these methods are apparent when 
one considers the huge economic losses which occur each year. 
It is therefore an object of the present invention to provide methods and 
compounds for inhibiting or preventing rust infections. 
It is another object of the present invention to provide methods and 
compounds which are safe, effective, and relatively inexpensive to use. 
It is a further object of the present invention to provide methods to 
reduce rust infection which can be used alone or in combination to 
increase effectiveness and decrease the probability of developing 
resistance to the compounds. 
It is a still further object of the present invention to provide methods 
and compounds which can be used topically, after infection occurs, to 
lessen reinfection. 
SUMMARY OF THE INVENTION 
Methods are provided to decrease leaf rust infections of plants, especially 
wheat, using inhibitors of the enzymes used by the leaf rust germ tube to 
ingest and metabolize components of epicuticular waxes, using ethylene or 
ethylene-like plant hormones to induce random appressoria formation at 
locations other than at the stomate, thus deterring successful infection, 
using genetic procedures to alter epicuticular wax composition, and/or 
using natural plant-growth regulators, precursors, or modifiers to modify 
epicuticular wax components. 
The major lipid classes present in the epicuticular waxes of wheat 
(Triticum aestivum L.) were separated and assayed for their utilization by 
wheat leaf rust (Puccinia recondita Rob. ex Desm.) spore germ tubes 
(WLRSGT) to produce .sup.14 CO.sub.2. Alkanes and esters were poorly 
utilized (39 and 31%, or less, respectively). -Diketones ( ), free fatty 
alcohols (FFAlc), HO- -diketones (HO- ), and free fatty acids (FFAc) were 
utilized between 71 and 100%. Wheat leaf rust infectivity correlated with 
and HO-contents with possible modification by FFAlc and FFAc contents. 
Inhibition can be achieved by alteration of the epicuticular wax 
composition to decrease the compounds utilized by the leaf rust germ 
tubes, especially the -diketones and HO- -diketones, using techniques such 
as selective breeding and genetic manipulation with screening for the 
production or non-production of particular compounds, as well as 
application of selected plant-growth regulators which modify epicuticular 
wax compounds. 
It was determined that ethylene induced appressoria formation by wheat leaf 
rust spore germ tubes. Maximum appressoria formation (50%) was attained at 
0.9 nmol/135 ml with decreased appressoria formation at ethylene 
concentration above and below this value. This method provides a further 
means to reduce successful infection.

DETAILED DESCRIPTION OF THE INVENTION 
Rust infection of cereal plants, including wheat, barley, oats, a variety 
of beans, corn and sorghum, is a major problem for farmers, with only 
expensive, ephemeral and prospective means available for dealing with the 
problem. The present invention presents alternatives having a number of 
advantages over the available means: they can be used after infection 
occurs to prevent an epidemic, they are effective and relatively safe to 
the farmer, compound is not lost into the surrounding soil, plants can be 
treated by aerial spraying, and variations and combinations of the 
compounds can be utilized to decrease the chance of resistance rust strain 
developing. 
One embodiment of the present invention uses enzyme inhibitors to decrease 
or block uptake of the epicuticular waxes by the migrating germ tube as it 
grows towards the stomate. The result is that fewer appressoria form and 
infection is therefore lessened. Compounds presently available are those 
which: 1) block the sulfhydryl groups on the germ tube enzyme(s), and 2) 
preferably do not penetrate the plant cell membranes. Compounds could also 
be developed using available technology to specifically inhibit the 
enzymes responsible for transporting and metabolizing the wax component, 
using the disclosed screening techniques. 
These compounds could be applied to plants in fields using sprays 
formulated with any applicable carrier suited to the 
hydrophilic/lipophilic balance of the chemical. Sprays should be topical 
and designed to produce minimum coverage, penetration and maximum 
efficacy. The sulfhydryl inhibitor must not penetrate the membranes while 
the proposed plant growth regulator must penetrate into the cell. These 
two compounds could be incorporated into a single spray treatment. 
Commercial applications could be by aerial or ground systems. 
Alternatively, plants can be selectively bred or genetically engineered to 
have an altered wax composition on the leaf surface. This must be 
contrasted with the methods now used to randomly generate and identify 
more resistant cultivars. Methodology and vectors are in use for inserting 
and modifying genes encoding the enzymes involved in wax synthesis. For 
example, Monsanto markets a vector for inserting genes into plants. The 
genetics and biosynthesis of plant epicuticular waxes are well 
characterized. See von Wettstein-Knowles, "Genetics and Biosynthesis of 
Plant Epicuticular Waxes", Advances in the Biochemistry and Physiology of 
Plant Lipids, Appelqvist and Lijenberg, ed. (Elsevier/North-Holland 
Biomedical Press 1979); von Wettstein-Knowles, Molec.gen.Genet. 144, 43-48 
(1976); Mikkelsen, Carlsberg Res.Commun. 44, 133-147 (1979); Netting, et 
al., Archives of Biochemistry and Biophysics 174,613-621 (1976); and von 
Wettstein-Knowles, Planta (Berl.) 106, 113-130 (1972). 
As an alternative to genetic manipulation of epicuticular wax composition, 
selected gibberellic acid derivatives induce alteration in epicuticular 
wax composition by increasing or decreasing the quantity of individual wax 
component classes. Gibberellic acids (GA) are known to induce increased 
stem length. It is shown herein that GA also induces decreased total wax 
content (ng/plant) with an accompanying increase in -diketone 
concentration. A degradation product of GA.sub.3 (GX) decreases -diketone 
content. Gibberic acid induces decreased -diketone content accompanied by 
an increase in total wax (ng/plant). The modification of leaf epicuticular 
wax composition by application of modified natural plant growth regulators 
(PGR) to attain a wax composition least favorable to the growth of the 
germ tube is demonstrated in the examples. These modified PGR do not have 
any other known influence on plant growth. 
In another embodiment of the present invention, plants were exposed to 
ethylene or an ethylene-like compound to induce random appressoria 
formation at locations not over stomates. The compound can be applied in 
aerosol, gas or sprayed in solution. This technique can be combined with a 
compound which inhibits transport and/or utilization of epicuticular wax 
components to provide maximum inhibition of rust infection, or in 
combination with compounds which induce modification of wax composition. 
Since infection requires formation of appressoria over stomates, these 
combinations are effective by: 1) inhibiting the utilization of the wax 
components present, 2) inhibiting the synthesis of additional components 
(i.e., -diketones) that are highly utilized by rust germ tubes, and 3) 
inducing appressoria formation randomly but not necessarily over the 
stomates, thus reducing infection. 
EXAMPLE I 
Demonstration of Wheat Leaf Rust Spore Germ Tube Utilization of 
Epicuticular Wax Components as an Energy Source 
Puccinia recondita Rob. ex Desm. urediniospores were sprayed onto wheat 
(Triticum aestivum L.) leaves and/or glass slides to which glycerol 
tri(1-.sup.14 C)palmitate or (1-.sup.14 C)linoleic acid had been 
previously applied and .sup.14 CO.sub.2 was trapped. Germinating 
urediniospores and/or sporeling germ tubes were determined to utilize 
extracellular lipids as an energy source. 
Wheat (Triticum aestivum L.) leaf rust (Puccinia recondita Rob. ex Desm.) 
urediniospores spores are very small and have limited food reserves having 
high energy content. Triacylgyceride content of stem rust (P. graminis f. 
sp. tritici) urediniospores declines during germination and germ tube 
development is dependent upon exogenous nutrients. 
In the past, germination and sporeling development of wheat leaf rust 
spores have been presumed to be independent of leaf epicuticular wax 
contents. It has now been observed that several cultivars express patterns 
in which infection is greatest at the base of the flag leaf blade and 
least at the tip and that this pattern is directly correlated with 
epicuticular wax contents of grass leaves which grow from a basal 
meristem. 
It was concluded from this observation, that some characteristic of wheat 
leaf epicuticular wax influenced spore germination and development. Since 
spores and sporelings utilize internal triglycerides as an energy source, 
it was decided to test whether they might also utilize portions of wheat 
leaf epicuticular waxes as an energy source. 
Urediniospores. 
Fresh wheat leaf rust urediniospores were supplied by the USDA-ARS National 
Cereal Rust Laboratory, St. Paul, Minn. Spores were suspended in deionized 
water to which two drops of the spreading agent, Triton X-100 were added. 
Germination counts were made on unstained material using the check slides. 
Culture Chamber. 
As depicted in FIG. 1, Warburg reaction chambers (135 ml) 10 were fitted 
with a perforated plexiglass support disk 12. Glass microscope slides 14 
were cut to lay on top of the support disk 12. Wheat leaves were treated 
with .sup.14 C containing lipid substrates and urediniospores were sprayed 
onto the leaves with an atomizer. The treated leaves were placed on the 
glass slides 14 which were positioned onto the support disk 12. 10 ml of 
water 16 was placed in the bottom of the flask 10. The reaction flask 10 
was sealed and .sup.14 CO.sub.2 was trapped in 10N KOH (3 ml 10N KOH on a 
piece of filter paper 18). After 24 hours at 28.degree. C. (water bath), 
the filter paper +10N KOH was recovered and inserted into 18 ml 
scintiverse. .sup.14 C was quantified by liquid scintillation spectroscopy 
(Beckman LS-100) for 50 minutes or 1% accuracy. Four replicates were 
utilized and data were statistically analyzed on a randomized block 
design. 
Substrates. 
.sup.14 C was supplied as: a) glycerol tri(1-.sup.14 C)palmitate (60 mCi/m 
mol) (5 Ci/treatment), or b) [1-.sup.14 C]linoleic acid (56.7 m Ci/mg) (5 
Ci/treatment). p-Chloromercuribenzenesulfonic acid (PCMBS) and 
dithiothreitol were applied at 1 and 10 mM concentrations, respectively. 
Substrate Utilization. 
Scanning electron photomicrographs of rust-inoculated wheat leaf surfaces 
showed what appeared to be germ tube tracks in the epicuticular wax, 
suggesting that germ tubes utilized epicuticular wax as a substrate. The 
presence of a living leaf is not necessary for urediniospore germination 
and germ tube development. However, the spores must contain a lipase to 
decompose the triglyceride so that fatty acids can be utilized as an 
energy source and some means of transferring the extracellular 
triglyceride and fatty acid into the germ tube must be present. 
Germinating spore and germ tubes utilization of extracellular triglycerides 
and fatty acids as energy sources is shown in Table 1. 
TABLE 1 
______________________________________ 
Conversion of glycerol tri(1-.sup.14 C)palmitate and 
(1-.sup.14 C)linoleic acid to .sup.14 CO.sub.2 by wheat leaf rust 
urediniospores on wheat leaves and glass slides 
Glycerol- (1-.sup.14 C) .sup.14 CO.sub.2 
tri(1-.sup.14) 
Linoleic Trapped 
palmitate acid Spores (dpm) 
______________________________________ 
Glass 
Slide 
- - - 50 e.sup.1 
- - + 175 d 
- + - 432 c 
+ - - 540 c 
+ - + 13373 a 
- + + 14649 a 
Leaves 
- - - 49 e 
- - + 497 c 
- - - 715 c 
+ - -- 210 d 
+ - + 5135 b 
- + + 7077 b 
______________________________________ 
.sup.1 Values in a column followed by the same letter are not 
statistically different at the 5% level. 
EXAMPLE II 
Inhibition of Wheat Leaf Rust Spore Germ Tube Utilization of Epicuticular 
Wax Components as an Energy Source 
It is known that enzymes containing extracellular sulfhydryl groups are 
responsible for transferring apoplastic sucrose into phloem sieve tube 
elements. These extracellular sulfhydryl groups are oxidized by PCMBS, 
which is membrane impermeant. Dithiothreitol (DTT) (or dithioerythritol) 
prevents the activity of PCMBS on the extracellular sulfhydryl groups. 
These combinations were, therefore, evaluated for germ tube utilization of 
[1-.sup.14 C]linoleic acid as an energy source. 
Impermeant PCMBS (1 mM) inhibited the conversion of [1-.sup.14 C]linoleic 
acid to .sup.14 CO.sub.2 by 77% (Table 2). DTT induced a 9-10% increase in 
[1-.sup.14 C]linoleic acid conversion to .sup.14 CO.sub.2. DTT reversed 
the inhibition by PCMBS so that spores treated with DTT PCMBS converted 
[1-.sup.14 C]linoleic acid into .sup.14 CO.sub.2 at a rate equivalent to 
83% of the conversion without either compound. 
TABLE 2 
______________________________________ 
Inhibition by p-chloromercuribenzenesulfonic acid 
(PCMBS) of wheat leaf rust spore conversion of (1-.sup.14 C) 
linoleic acid into .sup.14 CO.sub.2 and the reversion of that 
inhibition by dithiothreitol (DTT) 
[1-.sup.14 C]Linoleic .sup.14 CO.sub.2 
acid PCMBS DTT (dpm) 
______________________________________ 
+ - - 18133 b.sup.1 
+ + - 4126 d 
+ - + 19857 a 
+ + + 15076 c 
- + - 500 e 
- - + 475 e 
- - - 500 e 
______________________________________ 
.sup.1 Values in a column followed by the same letter are not 
significantly different at the 5% level. 
The conclusions drawn from these data are that the [1-.sup.14 -C]linoleic 
acid was transported into the germ tube by an enzyme containing 
extracellular sulfhydryl groups, PCMBS inhibited the activity of that 
enzyme, and DTT reversed the inhibition of PCMBS. 
It is therefore possible to inhibit germ tube growth (and subsequent 
infection) by exposing the infected leaf to compounds which inhibit 
sulfhydryl groups, taking care to select those which are not also toxic to 
the plant and meet EPA requirements. 
It should also be possible to inhibit germ tube growth by modifying the 
composition of the epicuticular wax of the host plant through: 1) PGR 
application, 2) genetic engineering, or 3) breeding with selection for 
altered epicuticular wax composition. von Wettstein-Knowles, p. 1-26 
"Genetics and biosynthesis of plant epicuticular waxes", in L. Appleguist 
and C. Lilyenberg, editors, Advances in the Biochemistry and Physiology of 
Plant Lipid, (Elsevier/North Holland Biomedical Press, New York, N.Y. 
1979). To facilitate this process, the composition and utilization of the 
epicuticular waxes by germ tubes were therefore determined. 
EXAMPLE III 
Utilization of Extracellular Lipids by Germinating Puccinia recondita 
Urediniospores 
.sup.14 CO.sub.2 was utilized to label the epicuticular wax components of 
wheat (Triticum aestivum L., cvs. Coker 983, Florida 301, Red Bobs, and 
Hunter). The major lipid classes were separated and assayed for their 
utilization by wheat leaf rust (Puccinia recondita Rob. ex Desm.) spore 
germ tubes (WLRSGT) to produce .sup.14 CO.sub.2. Alkanes and esters were 
poorly utilized (39 and 31%, or less, respectively). -Diketones (), free 
fatty alcohols (FFAlc), HO- -diketones (HO- ), and free fatty acids (FFAc) 
were utilized between 71 and 100%. Wheat leaf rust infectivity correlated 
with and HO- contents with possible modification by FFAlc and FFAc 
contents. -Diketone contents increased from the tip to the base of Coker 
983 flag leaves. Esters were most concentrated at the tip and least 
concentrated at the mid- and base-sections. 
It was observed that wheat leaf rust spores infect the base of the flag 
leaf of some slow-rusting cultivars more heavily than the tip. This 
observation, in combination with the determination that wheat leaf rust 
spore germ tubes (WLRSGT) utilize epicuticular lipids as an energy source 
and that utilization is inhibited by p-chloromercuribenzenesulfonic acid 
(PCMBS) which is known to inhibit extramembrane enzymes containing 
sulfhydryl (SH) groups, and scanning electron photomicrographs providing 
evidence of intimate contact between germ tubes and epicuticular waxes 
(FIGS. 2 and 3) led to the hypothesis that germ tube growth was dependent 
on the availability of certain components of the epicuticular waxes. 
Wheat and barley (Hordeum vulgare L.) epicuticular waxes are known to 
contain -diketones and HO- -diketones. These compounds are comparatively 
unique to cereals. Studies were therefore conducted to assess whether or 
not WLRSGT utilized other components of the leaf waxes, in addition to 
triglycerides and linoleic acid, as energy sources. 
Methods and Materials: 
Wax .sup.14 C Content: Three wheat plants/pot (4 pots/exposure) were grown 
in soil in the greenhouse to the boot stage (Feekes scale stage 10, that 
stage where the wheat spike or head is enclosed within the flag leaf 
sheath tube, just prior to the heading. cf. D. R. Tottman and R. J. 
Makepeace, "An Explanation of the Decimal Code for the Growth Stages of 
Cereals." Am. Appl. Biol. 93, 221-234 (1979)). The plants were placed in a 
90 cm diameter clear polyethylene tube without being exposed to direct 
sunlight. The tube was sealed and 2 Ci .sup.14 CO.sub.2 was released from 
Na.sub.2.sup.14 CO.sub.3 (10 mCi/mM) by concentrated HCl. After 18 h of 
exposure to .sup.14 CO.sub.2, the plants were removed from the 
polyethylene tube and epicuticular waxes were recovered by chloroform 
extraction using the method of Martin, J.Sci.Food Technol. 11, 635-640 
(1960). The solvent was evaporated to dryness and 10 ml CHCl.sub.3 was 
utilized to liquify the residue. Lipid classes were separated by 
thin-layer chromatography according to the method of Tulloch and Hoffman, 
Phytochem. 10, 871-876 (1971) utilizing 0.25 mm silica gel g and 
chloroform:ethanol (99:1, v,v) as a developing solvent. The separated 
lipid classes were individually eluted into chloroform and concentrated 
with N.sub.2. Four cultivars of wheat (Coker, 983, Florida 301, Red Bobs, 
and Hunter) were treated individually. 
Lipid Substrate Utilization: As described previously with respect to FIG. 
1, growth chambers were 135 ml Warburg flasks fitted with a perforated 
plexiglass disk. Ten ml water was added to the bottom of the flask and the 
side arm contained fluted (6 cm.times.4 cm) Whatman No. 1 filter paper +3 
ml 10% KOH. Fifty 1 of a lipid were spread evenly over the surface of a 
glass slide (37.5 mm.times.25 mm) and the solvent was evaporated. Thirty 
mg urediniospores were mixed in 10 ml deionized water +2 drops Triton 
X-100 in an atomizer. After vigorous shaking, the spore suspension was 
evenly sprayed onto the glass slides which were then placed on the 
perforated plexiglass support. The chamber was sealed, placed in a 
28.degree. C. water bath, and the water bath was covered with black 
polyethylene. After an 18 h incubation, the filter paper was removed and 
inserted into a scintillation vial with 18 ml Scintiverse. .sup.14 
CO.sub.2 content was assayed by liquid scintillation spectrometry (Beckman 
LS-100) for 50 min or 1% accuracy after 24 h in the dark for fluorescence 
decay. Backgrounds were subtracted from the total DPM. Assays of each 
lipid class were conducted four times. 
Coker 983 Wax Content: Coker 983 flag leaves, 19-20 cm long, were harvested 
from field-grown plants and weighed. Epicuticular waxes were extracted 
from three cm sections cut from the tip, middle, and base of the leaves in 
chloroform and the extract reduced to 10 ml. -Diketone content was 
quantitated by spectrophotometry (Beckman DB-GT) at 273 nm in quartz 
cuvettes with E.sub.1 cm.sup.1% 250. 
The lipid classes of the remainder were quantitatively separated by TLC, as 
described above. Internal standards (1 mg) (heptadecanoic acid, 
heptadecanol, or n-docosane) were added as required and fatty acid methyl 
esters or fatty alcohol formyl esters were prepared as described by 
Wilkinson and Mayeux in Bot.Gaz. 148, 12-16 (1987). Esters were quantified 
by gas-liquid chromatography (Hewlett-Packard 5751A) using a dual FID 
detector and 0.32 cm diameter stainless steel columns 305 cm long filled 
with 5% OV-101 on 80/100 mesh Anakrom ABS. The column was programmed at 
4.degree. C./min with a 10 min upper limit hold. Detector and injector 
port temperatures were 380 and 370.degree. C., respectively. Five 
replications were used throughout. Data were converted to g/g fresh 
weight. 
Incorporation of .sup.14 CO.sub.2 into epicuticular wax lipid classes 
(Table 3) shows alkane contents vary between cultivars (Coker 983 
Florida 301 Red Bobs=Hunter, 64.63, 41.31, 20.50, and 19.16 weight %, 
respectively). Ester .sup.14 C content varied between 27.52 and 10.24%. 
-Diketone .sup.14 C content was approximately inverse to the alkane 
contents (Coker 983 .sub. Florida 301=Red Bobs .sub.- Hunter; 5.33, 16.02, 
15,82, 49.35 weight %, respectively). Various patterns were found for free 
fatty alcohols, HO- -diketones, and free fatty acids. The cultivars 
differed widely in their ability to incorporate .sup.14 CO.sub.2 into 
epicuticular wax components (Total DPM/ml wax). The patterns shown in 
Table 3 are based on the assumption that the composition of the 
epicuticular wax follows the same pattern as the .sup.14 CO.sub.2 
application, at the time of .sup.14 CO.sub.2 treatment. 
TABLE 3 
______________________________________ 
.sup.14 C Content of Epicuticular Wax Lipid Classes 
Coker Florida Red 
983 301 Bobs Hunter 
DPM DPM DPM DPM 
Lipids (%) (%) (%) (%) 
______________________________________ 
Alkanes 6982 3519 470 397 
(64.63) (41.31) (20.50) 
(19.16) 
Esters 2973 872 391 366 
(27.52) (10.24) (16.62) 
(17.69) 
Diketones 575 1364 362 1022 
(5.33) (16.02) (15.82) 
(49.35) 
Free Fatty Alcohols 
179 1196 778 162 
(1.66) (14.04) (33.92) 
(7.82) 
Diketones 32 502 170 81 
(0.30) (5.90) (7.44) (3.91) 
Free Fatty Acids 
59 1064 130 43 
(0.55) (12.49) (5.67) (2.08) 
Total (DPM/ml) 
10800 8517 2292 2072 
______________________________________ 
Lipid Substrate Utilization: WLRSGT utilized the .sup.14 C-lipid classes to 
produce .sup.14 CO.sub.2 at different rates, as demonstrated in Table 4. 
Although, alkanes and esters were poorly utilized as energy sources to 
produce .sup.14 CO.sub.2, the other four classes of lipid constituents in 
the epicuticular waxes were readily utilized to produce .sup.14 CO.sub.2. 
TABLE 4 
______________________________________ 
Utilization of .sup.14 C-Lipid Classes Utilized to 
Produce .sup.14 CO.sub.2 by Wheat Leaf Rust Spore Germ Tubes 
Coker Florida Red 
983 301 Bobs Hunter 
Lipids % .sup.14 C applied 
______________________________________ 
Alkanes 38.9 1.0 0.3 23.8 
Esters 30.8 4.5 6.0 13.6 
Diketones 70.8 62.8 21.8 72.0 
Free Fatty 98.0 99.0 99.0 99.0 
Alcohols 
Diketones 98.0 99.0 99.0 98.0 
Free Fatty Acids 
99.0 98.0 76.9 99.0 
Acids 
______________________________________ 
Comparison of wheat leaf rust infection with various combinations of 
-diketones, free fatty alcohols, HO- -diketones, and free fatty acids 
(Table 5) shows that combinations of these constituents, generally, 
correlate with infectivity. However, -diketone+HO- -diketone levels have a 
very close correlation with relative infection. 
TABLE 5 
______________________________________ 
Comparison of Wheat Leaf Rust Infectivity 
Between Wheat Cultivars and the .sup.14 C Contents 
of Selected Epicuticular Wax Components 
Leaf 
Cultivar Rust % A B C D 
% % .sup.14 C Content 
______________________________________ 
Florida 301 
10 12.4 2.6 2.3 5.6 
Coker 983 40 7.8 2.5 2.1 10.2 
Hunter 90 63.1 13.8 9.9 23.2 
Red Bobs 100 62.9 46.7 42.7 53.2 
______________________________________ 
A =Diketones + Free Fatty Alcohols + HO--Diketones + Free Fatty Acids 
B = Free Fatty Alcohols + HO--Diketones + Free Fatty Acids 
C = Free Fatty Alcohols + Free Fatty Acids 
D =Diketones + HO--Diketones 
Coker 983 Wax Content: Wheat leaf rust infection severity increases from 
the tip to the base of the flag leaf in some cultivars. The -diketones and 
HO- -diketones are intimately associated with infection efficiency and 
utilization of the wax as an energy source. Contents of these constituents 
would be expected to increase from the tip of the leaf toward the base, as 
shown in Table 6. Additionally, there are minor decreases in total alkane 
content from the tip to the base (Table 7), as compared to .sup.14 
CO.sub.2 incorporation into alkanes, shown in Table 3, and significant 
decreases in ester content from the tip to the base of the flag leaf 
(Table 7). -diketone content (in g/g FW) more than doubled form the tip to 
the base (Table 7). 
TABLE 6 
______________________________________ 
Diketone Content of Coker 983 Flag Leaves 
Diketones 
DiketonesO-- 
leaf portion g/g FW 
______________________________________ 
Tip 1.4 c 0.7 b 
Mid 3.1 b 0.4 c 
Base 5.4 a 1.0 a 
______________________________________ 
TABLE 7 
______________________________________ 
Coker 983 Flag Leaf Epicuticular Wax Contents 
Leaf Mid- Leaf 
Base Leaf Tip 
Lipids wt. % 
______________________________________ 
Alkanes 3.10 6.74 4.11 
Esters 13.83 12.81 46.53 
Diketones 57.42 57.43 23.38 
Free Fatty Alcohols 
10.53 14.06 9.50 
Diketones 10.99 7.93 11.00 
Free Fatty Acids 
4.13 1.03 5.58 
Total (g/g FW) 
9376.5 5349.4 6004.4 
______________________________________ 
Wheat leaf rust urediniospores are very small with very limited internal 
food reserves usually in the form of triglycerides. Utilization of an 
external substrate source is beneficial to the organism during the 
infection process. Adaptation to utilization of the external food sources 
present on host species most conducive to growth and infection by the rust 
species provides it with a real selective advantage. -Diketones have been 
analyzed from several wheat cultivars and found to have a very limited 
range of constituents. Adaptation to these relatively unique energy 
sources would serve as a real and positive selection mechanism in the 
WLRSGT as it grows laterally across the wheat leaf until it finds a stoma 
where appressorium formation and penetration into the leaf occurs. Thus, 
under standard conditions, alterations in wax composition that are 
beneficial to WLRSGT would increase the range of the germ tube and, 
presumably, the relative chances of successful infection. Conversely, 
development of cultivars having fewer favorable wax components offers 
another means to reduce damage from rust infection. Such relatively 
complex changes in the nature of the leaf surface may pose a more 
difficult puzzle to the rust, making this a long-lasting form of partial 
resistance of potential value in supplementing other, more conventional 
forms. 
EXAMPLE IV 
Ethylene Induction of Appressoria Formation by Wheat Leaf Rust (Puccinia 
recondita Rob. ex Desm.) Spore Germ Tubes 
Wheat leaf rust urediniospores produce germ tubes which grow laterally 
across the wheat leaf surface. When the germ tube encounters a stomate, an 
appressorium is formed. Appressoria formation has been induced by 
acrolein, contact with stomate ridges, teflon, and an "effluvium" from 
stomates. 
Ethylene is a gaseous plant growth regulator produced by plants, which 
induces a variety of responses including cessation of horizontal growth. 
Plant responses to ethylene depend highly upon concentration, tissue, and 
species. Ethylene was evaluated to determine the influence of ethylene on 
leaf rust spore germ tube elongation and appressoria formation. 
Methods and Materials. 
Culture: Using a 135 ml Warburg flask fitted with a perforated polystyrene 
disk+10 ml deionized water (as described before with reference to FIG. 1), 
wheat leaf rust uredispores (10 ml water+2 drops Triton X-100) were 
sprayed onto glass microscope slides (37.times.25 mm) coated with 
petrolatum. The slides were prepared by heating on a slide warmer 
(65.degree. C.) for 24 hours to produce an even petrolatum covering 
without ridges. The slides were placed into the chambers. The chambers 
were maintained at 28.degree. C. in a water bath and darkened with a 5 mil 
black polyethylene cover. 
After an 18 h germination, ethylene was inserted into the chambers, the 
chambers were resealed and covered, and development was continued for 7 
hours. Fifty mg urediniospores per 10 ml water were used in studies 1 and 
2, and 100 mg urediniospores/10 ml water were used in study 3. Each test 
was conducted in quadruplicate. 
Urediniospore Counting: Slides were dipped for 8 seconds in acid fuchsin to 
stain germinated structures. Counts were made at 430x of germ tubes and 
germ tubes with appressoria. Only those which could be linked to a spore 
were included. 
Study 1: Ethylene was inserted after 4 hr growth at concentrations 100 
nmol/135 ml. All spore germ tube growth ceased under these conditions, 
although untreated germ tubes grew normally with 5% appressoria formation. 
The conclusion was therefore that these ethylene concentrations were too 
high and initial spore tube growth was insufficient. 
Studies 2 and 3: Urediniospore germ tube appressoria formation was induced 
50% by 0.9 nmol/135 ml ethylene. Increasing or decreasing the 
concentrations of ethylene significantly decreased the percentage of 
germtubes that developed appressoria. 
Stem rust produced appressoria at about 25% at the same ethylene 
concentration. 
EXAMPLE V 
Control of Wheat Leaf Epicuticular Wax Composition by Gibberellic Acid 
(GA.sub.3) and its Degradation Products 
Wheat leaf rust urediniospores produce germ tubes which ingest epicuticular 
wax lipids as an energy source. Within the total waxes, two major 
biosynthetic schemes are know. One produces alkanes, esters, free fatty 
alcohols and free fatty acids, while the other produces the -diketone 
series of compounds. Gibberellic acid (GA.sub.3) is known to induce the 
synthesis of glyoxysomes at specific plant growth stages which utilize the 
fatty acid base lipids to produce Ac-CoA. 
Exogenous application of GA.sub.3, a natural PGR, was therefore tested for 
alteration of the epicuticular wax composition of wheat leaves. 
Methods and Materials: 
Twenty-five wheat (cv Stacy) seeds were planted in sand in 
10.times.10.times.10 cm pots and the initial watering was with 100 ml 
Hoagland and Aron complete mineral nutrient solution. After 10 days, the 
plants were sprayed with GA.sub.3 -methyl ester (0, 0.1, 1.2, 5, or 100M) 
in water (40 gpa) containing 3 ml Chem-nut oil per 250 ml water. Waxes 
were extracted in CHCl.sub.3 and evaporated to dryness. The total wax (mg) 
was determined by weight and the -diketone content was determined 
spectrophotometrically. 
In succeeding studies, gibberic acid (G) or a water soluble GA.sub.3 
degradation product (GX) was substituted for GA.sub.3. 
GA.sub.3 -Methyl Ester: GA.sub.3 -Methyl Ester induced a decreases in total 
wax (mg/plant) concentration without influencing -diketone concentration, 
as shown in Table 8. 
TABLE 8 
______________________________________ 
Influence of GA.sub.3 on growth and epicuticular wax quantity 
and composition of wheat (Triticum aestivum L. cv Stacy). 
GA.sub.3 -Methyl Ester ( M) 
Days 0 0.1 1.2 5.0 100 
______________________________________ 
(Height) 
0 29.6 
0.sup.+2 
27.0 
3 28.8a.sup.1 
29.8a 
29.8a 28.8a 
28.6a 
7 31.0c 32.0c 
36.0b 37.0b 
39.0a 
Fresh 0 227 
Weight 0.sup.+ 
209 
(mg/plant) 
3 260c 271b 291a 268c 262c 
7 312d 325c 329b 357a 319cd 
Total Wax 
0 8b 
(mg/plant) 
0.sup.+ 
8b 
3 11a 7b 12a 4ed 2e 
7 6bc 5c 4cd 7b 5c 
diketone 
0 11.3d 
(%) 0.sup.+ 
10.1de 
3 9.4e 15.7e 
11.0d 19.4a 
17.5b 
7 19.8a 20.6e 
22.9a 19.5a 
21.4a 
Total Wax- 
3 9 7 10 5 0 
Diketones 
7 4 4 3 5 2 
______________________________________ 
.sup.1 Values on a line followed by the same letter are not significantly 
different at the 5% level. 
.sup.2 Plants which received the waterChemnut oil spray without GA.sub.3. 
Each value is the average of five replicates. 
Gibberic acid (G) induced an increased total wax minus -diketones 
concentration but not an accompanying increase in -diketone concentration. 
As a result, the % -diketone content was drastically decreased, as 
demonstrated in Table 9. 
TABLE 9 
______________________________________ 
Influence of gibberic acid-methyl ester on the 
epicuticular wax composition of wheat (Triticum aestivum 
L. cv Stacy) leaves seven days after application. 
Gibberic Acid-Methyl Ester ( M) 
Constituent 0 0.1 1.2 5.0 100 
______________________________________ 
Total Wax- 13d.sup.1 
249c 362b 307bc 581a 
Diketones 
(.DELTA. g/plant) 
Diketones 25a 17b 16b 11c 9c 
(%) 
______________________________________ 
.sup.1 Values in a line followed by the same letter are not significantly 
different at the 5% level. 
Each value is the average of five replications. 
Neither G nor GB influenced plant elongation or increase in fresh weight. 
Induction of the synthesis of isocitric-lyase, a critical enzyme in 
glyozysomes involved with the -oxidation of fatty acids, by GA.sub.3 is 
shown in Table 10. 
TABLE 10 
______________________________________ 
Influence of gibberellic acid (GA.sub.3) on the isocitric 
lyase content of wheat (Triticum aestivum L. cv Stacy) 
leaks at 3- and 7-days after GA.sub.3 application. 
Isocitric GA.sub.3 -Methyl Ester ( M) 
Lyase Days 0 0.1 1.2 5.0 100 
______________________________________ 
nmol 3 23.4a.sup.1 
18.8a 
22.6a 
23.7a 21.4a 
product 7 7.5c 17.1a 
15.5a 
15.5b 15.7b10.7c 
min 
mg-protein 
nmol 3 1.0a 1.3a 1.3a 1.3a 1.1a 
product 7 2.6c 3.8b 3.7b 3.7b 4.3a 
min 
g Fresh Weight 
______________________________________ 
.sup.1 Values in a line followed by the same letter are not significantly 
different at the 5% level. 
Each value is the average of five replications with triplicate assays per 
relication. 
TABLE 11 
______________________________________ 
Influence of a GA.sub.3 degradation product on the growth 
and epicuticular wax quantity and composition is seedling wheat 
(Triticum aestivum L. cv Stacy) grown in nutrient solution 
GX ( M) 
Days 0 1 10 100 
______________________________________ 
Height 0 26.0 
(cm) 3 27.6a.sup.1 
27.0a 28.2a 28.2a 
7 34.4a 32.2a 33.0a 33.0a 
Fresh 0 211 
Weight 3 297a 287a 309a 282a 
(mg/plant) 
7 484a 428b 420b 432b 
Total wax 
0 469 
(mg/plant) 
3 495a 301b 191c 223c 
7 484a 428b 420b 432b 
Diketone 0 6 
(mg/plant) 
3 54a 15b 6b 6b 
7 57a 12b 9b 7b 
Diketone 3 100a 19.2b 0.2e -0.6e 
(%) 7 100a 17.4b 8.8e 5.0d 
______________________________________ 
This study demonstrates the alteration of the composition of epicuticular 
waxes by the topical application of natural plant-growth regulators or 
their precursors or degradation products. With the latter two compounds, 
no other response by plants is known. 
Modifications and variations of the present invention, methods and 
compounds to inhibit leaf rust infections, will be obvious to those 
skilled in the art from the foregoing detailed description of the 
invention. Such modifications and variations are intended to come within 
the scope of the appended claims.