Antifungal Proteins and Other Mechanisms in the Control of
Sorghum Stalk R ot and Grain Mold
R. D. Waniska,*,†R. T. Venkatesha,‡A. Chandrashekar,‡S. Krishnaveni,§F. P. Bejosano,†
J . J eoung,§J . J ayaraj,§S. Muthukrishnan,§and G. H. Liang#
Cereal Quality Laboratory, Texas A&M University, College Station, Texas 77843-2474; Central Food
Technological Research Institute, Mysore, India; and Departments of Biochemistry and Agronomy,
Kansas State University, Manhattan, Kansas 66506
Research on antifungal proteins and other mechanisms that provide the biochemical basis for host-
plant resistance tostalk rot and grain molds is reviewed in this paper. Stalk rot caused by Fusarium
species leads tosubstantial yield loss due topoor grain filling and/or lodging. A transgenic sorghum
expressing high levels of chitinase exhibited less stalk rot development when exposed to conidia of
F. thapsinum. Grain mold of sorghum is associated with warm humid environments and results
from colonization by several fungi (F. thapsinum, Curvularia lunata, and Alternaria alternata) of
thedeveloping caryopsis. Theroles of several biochemical mechanisms (tannins, phenolic compounds,
red pericarp, proteins, hard endosperm, and antifungal proteins) on grain mold resistance are
discussed. Resistancemechanisms related tothesecompounds appear tobeadditive, and pyramiding
of genes is a feasible approach to limit grain deterioration. Several experimental approaches are
proposed to extend current findings.
K eywords: Antifungal proteins; grain mold; stalk rot; fungal pathogens
Sorghum [Sorghum bicolor (L.) Moench] is used for
fodder, feed, food, and beverage. Sorghum is cultivated
in tropical and temperate zones and is exposed to a
broad range of diseases. Sorghum plants are attacked
by fungal, bacterial, and viral pathogens causing root,
stalk, foliar, panicle, and caryopsis diseases. We will
review biochemical mechanisms to control fungal dis-
eases of the root and stalk before turning to the fungal
diseases of the caryopsis.
FUNGAL ROOT AND STALK ROT OF SORGHUM
Fungi cause many severe diseases, including root and
stalk rot as a result of infection by Fusarium monili-
forme, Fusarium thapsinum, or Colletotrichum gramini-
cola, seedling diseases induced by Pythium sp., foliar
diseases such as sooty stripe incited by the causal
pathogen Ramulispore sorghi, and head smut caused
by Sporisorium reilianum. Fusarium stalk rot caused
>$80 million lost grain yield in 1996 in Kansas. Yield
loss duetostalk rot is attributed directly tolower kernel
weight and weakened peduncles or indirectly tolodging
and stalk breakage. Fusarium sp. are spread by wind,
rain, machinery, and insect damage. The fungus pen-
etrates plants directly or via natural openings, such as
stomata. It survives as spores or resting structures in
soil or plant debris or as hyphae within living plants.
Pathogenesis-related (PR) roteins are inducible plant
defenses that restrict the spread of the pathogen in
incompatible interactions and allow for systemic ac-
quired resistance. Most research has been carried out
with leaves and stalks where there is induction of
synthesis mechanisms. A review of such studies made
mostly on tobaccowas thoroughly covered by Linthorst
(1). Many members of this group of proteins have in
vitro antifungal activity and selectively target cellular
components of the pathogen. Included in this group are
chitinases and ?-1,3-glucanases, which attack the cell
walls of fungi, and thaumatin-like proteins (TLP) that
affect the permeability of fungal membranes. Genes or
cDNAs for these proteins have been isolated from
cereals including rice, barley, and wheat (2-4). Several
major cereals (rice, maize, wheat, and barley) havebeen
transformed successfully by both biolistic and Agrobac-
terium-mediated techniques (5). Constitutive over-
expression of these genes in transgenic rice and wheat
plants results in improved resistance to fungal patho-
gens (6, 7).
Sorghum has been recalcitrant to transformation;
however, a gene coding for a rice chitinase was incor-
porated into an elite sorghum inbred line, Tx430, by a
biolistic transformation protocol. Six primary transgenic
plants were obtained and were shown to contain the
transgene by Southern blot analysis (8). The chitinase
transgene was inherited in a Mendelian fashion by
progeny from these primary transgenic plants and was
expressed in the T1 and T2 generations. We have
identified one transgenic line that does not segregate
for the transgene locus. The chitinase transgene con-
tinues to be expressed in T2 and T3 generation plants
as measured by western blot analysis of leaf extracts
using a chitinase antibody. Plants from the T2 and T3
generations were tested for resistance to infection by
inoculating the stalks of mature plants with conidia of
F. thapsinum (112 conidia mL-1). The development of
stalk rot symptoms was significantly reduced in trans-
†Texas A&M University.
‡Central Food Technological Research Institute.
§Department of Biochemistry, Kansas State University.
#Department of Agronomy, Kansas State University.
4732J. Agric. Food Chem . 2001, 49, 4732−4742
10.1021/jf010007f CCC: $20.00 ©2001 Am erican Chem ical Society
Published on Web 09/27/2001
genic sorghum plants with higher expression of rice
chitinase, whereas plants with low or no expression of
rice chitinase showed normal disease progression. The
constitutive overexpression of rice chitinase in sorghum
plants seems to offer protection against the stalk rot
Alternatives to the biolistic procedure have been
explored because the biolistic procedure often leads to
a high copy number of transgenes, which sometimes
result in genesilencing in advanced generation progeny.
The gene encoding a rice TLP was introduced into
inbred line C-401 using the Agrobacterium strain LBA
4404. Several independent transformants appeared to
have one or a few copies of the tlp gene with good
expression of TLP as shown by western blot analysis of
leaf extracts using an antiserum.
More TLP (such as osmotin) protects the plants
against fungal pathogens as well as against salt and
drought stress (9). The T2 generation transgenic sor-
ghum plants with a high level of rice TLP were tested
for resistance towater stress. Preliminary data indicate
that the transgenic plants (1-month-old seedling) with
the tlp gene also show tolerance to water stress.
FUNGAL CARYOPSIS DISEASES OF SORGHUM
Grain mold of sorghum results from colonization of
fungi (F. thapsinum, C. lunata, and A. alternata) of the
developing grain and is associated with warm, humid
environments during caryopsis development. Themajor
colonizing fungi also produce mycotoxins, which are
harmful to humans and livestock (10-12). Fungal
growth causes surface discoloration, initially, and con-
tinues, provided warm moist conditions, tobreak down
the components of the grain. These changes decrease
milling and processing yields and quality of sorghum
for feed or food. Plant breeding efforts have been only
partially successful (13), whereas control of grain mold
by using fungicides and crop management strategies is
beyond the means and abilities of many farmers.
Plant traits such as panicle shape, plant height, and
glume structure have been associated with grain mold
resistance (14, 15). Caryopsis traits such as endosperm
hardness, a pigmented testa layer, and red pericarp
color are correlated with grain mold resistance (16-19).
Although many sorghums with tannins and higher
levels of phenol-based pigments are more resistant to
molds, these compounds cause dark colors, astringency,
and/or decreased nutritional value in foods or feeds (20,
21). Accordingly, sorghums for food use are grown in
environments that are not conducive to deterioration,
with a correspondingly lower yield, or cultivars resistant
to grain mold are selected, which yield less desirable
food. Hence, grain mold resistance is necessary in food-
A review of strategies used by the sorghum grain to
combat infestation by fungi is presented here. Included
are discussions on fungal colonization, caryopsis struc-
ture, phenolic compounds, and antifungal proteins
(AFP). We hope our observations lead to the develop-
ment of newer strategies in thefight against grain mold.
F ungal Invasion. Forbes et al. (12) reviewed colo-
nization events in mold-resistant and -susceptible cul-
tivars. During the initial period of flower invasion, C.
lunata can infect the apical part of the ovary wall from
thecolonized lemma, palaea, lodicules, filaments, pollen
grain, and decaying style (22). Mycelium penetrates the
pericarp and ramifies through the cross and tube cells
within 5-10 days. Generally, the pericarp is not colo-
nized. The placental sac offers a niche for fungal growth
that subsequently invades the endosperm and some-
times the embryo as well. F. thapsinum appears later
and grows beneath the pericarp and then attacks the
floury endosperm. Differences in infection patterns of
C. lunata and F. thapsinum are currently being studied
(L. Prom, unpublished data).
Mycotoxins. Mycotoxins are less of a problem in
sorghum than in maize. Lower levels of fumonisin were
found in sorghum (0.7-36.1 ppm) than in maize (5-
4000 ppm) (23, 24). Normal sorghums used to prepare
Indian foods contain very little fumonisin A or B, which
are produced by F. thapsinum (25). Leslie et al. (26)
reported that the F mating population of the fungus,
which is dominant on sorghum, produces little or no
fumonisin, whereas the A mating population, which is
abundant on maize, produces more fumonisin. Klittich
et al. (27) observed that F. thapsinum does not produce
Aflatoxins are not found at significant levels in
sorghum brought directly in from the field (G. Odvody,
personal communication). High levels of mycotoxins,
including aflatoxins, are associated with high-moisture
storage of sorghum and other cereals. Maize may have
excessive levels of aflatoxins, whereas sorghum is afla-
toxin-freein similar drought-proneenvironments. Some
sorghums contain another type of toxin, deoxyvarenol,
which is an emetic and toxic torats (28-30). Alternaria
also produces toxins, which are confused with metabo-
lites from other fungi (31). Ergosterol levels correspond
to nonspecific mold colonization of sorghum and have
been used to monitor grain molding, but they have not
been linked to animal health problems (32).
Ergot, Claviceps africana, has recently infested sor-
ghums in the Western hemisphere (33). The sclerotia
contain alkaloids that may cause problems when fed to
Grain Structure and Development in Sorghum.
The structure of the mature grain of sorghum, like all
cereals, is composed of the triploid storage endosperm
and diploid zygotic embryo surrounded by the pericarp
(a maternal tissue) and testa. The testa in some
sorghum species is pigmented and contains condensed
tannins. The thickness of the testa varies from 8 to 40
µm (35), with the thickest area being below the style
and the thinnest on the side of the kernel. Many
sorghums containing high levels of tannins have a high
degree of resistance to molds (36); however, most
commercial hybrid and feed- and food-type sorghums
are virtually tannin free (37). The pigmented testa is
seen as a dark layer between the light endosperm and
the pericarp when the caryopsis is scraped to remove
The endosperm, the bulk of the caryopsis, is inside
the pericarp and testa (Figure 1). The first layer, the
rectangular aleurone cells, have thick walls and contain
oil and protein bodies. Then, three layers of starchy
endosperm cells can be recognized. The peripheral area
is inside the aleurone cells and comprises several layers
of dense cells that are rich in protein but contain only
small starch granules. Inside this area is the corneous
(horny) area, which contains angular starch granules
embedded in a continuous protein matrix. In the center
is a white, opaque, floury area in which the protein is
loosely packed with air spaces and the starch granules
are more spherical in shape (38).
SorghumAntifungal Proteins in GrainJ. Agric. Food Chem ., Vol. 49, No. 10, 2001 4733
McDonough and Rooney (39) reported that the peri-
carp is completely formed 6-9 days after anthesis (DAA)
and, thereafter, begins to compress. Cells in the testa
layer are apparent 3-6 DAA, whereas the aleurone
layer took longer (6-12 DAA) to develop. Ovary walls
contain simple and compound starch granules at an-
thesis. Simple starch granules and protein bodies begin
to develop in endosperm cells by 3-6 DAA. Protein
bodies at this stage were covered by a thin filamentous
webbing, which later develops intoa distinctive protein
matrix. Shull et al. (40) noted that hard-endosperm
sorghums that are more resistant to molds deposit
protein much earlier than do floury-endosperm sor-
ghums (Figure 1).
Grain T exture and R esistance to Grain Molds.
The proportions of the outer translucent layer (also
called glassy, horny, vitreous, or corneous) and theinner
opaque, white area (also called soft or floury) vary
among cultivars (41). Grain hardness is related to the
relative areas of corneous and floury endosperm. En-
dosperm hardness attenuates growth of grain mold (16-
19, 42). Molds more quickly deteriorate the endosperm
structure of susceptible cultivars compared toresistant
cultivars, even though they may have similar endo-
sperm hardnesses in a mold-free environment (43).
A clue to the resistance mechanism related to en-
dosperm texture comes from studies of mutant lines of
maize. The opaque2 mutation increases lysine but has
a soft texture, susceptible toinfection by molds (44, 45),
and reduced levels of R-zeins and ribosome-inactivating
protein (RIP) (37). A DNA-binding protein encoded by
the opaque2 locus (46-48) regulates the levels of both
γ-zein and RIP. The homologous RIP from barley has
antifungal properties (49), whereas Dowd et al. (50)
suggests that maize RIP may be insecticidal. Decreased
levels of RIP may contribute to susceptibility to molds.
The hard-textured types of opaque2 maize contain the
same lower levels of γ-zein, as do the unmodified
opaque-2 lines, whereas their γ-zein levels areincreased
The corneous endosperm of sorghum is enriched in
kafirins, especially γ-kafirins (42, 53, 54). Theγ-kafirins
contain more cysteine and form extensive intra-
chain disulfide bonds, which may contribute both to
hard texture and to resistance to fungal infection (55,
56). The prolamins in the protein bodies are remark-
ably resilient to proteolysis by a fungal protease until
their disulfide bonds are reduced (Mazhar and Chan-
drashekar, unpubublished results).
The cell wall composition also varies between the
corneous and floury endosperm and between hard and
soft grains (57, 58). Because the endosperm structure
is modified during fungal colonization of the caryopsis,
cell wall structure and composition need to be investi-
gated to increase resistance to grain molds.
A combination of factors has recently been shown to
correspond tograin mold resistance(59). Sorghums with
red pericarp and/or tannins (Table 1) did not have to
have a hard endosperm toexhibit resistance, but white
pericarp sorghums needed a hard endosperm before
some level of resistance was observed. Otherwise,
sorghums with low and medium endosperm hardness
exhibited the most deterioration. Harder grain had
lower mold ratings when stressed with sprinkling or
inoculation with fungal pathogens.
T annins and Phenolic Compounds. Most sor-
ghums donot contain tannins but all sorghums contain
phenols and most contain flavonoids. Sorghums are
classified as type I (no tannins), type II (tannins in
pigmented testa), or typeIII (tannins in pigmented testa
F igure 1. SEM photomicrograph montage of mature hard (top, Mishimba) and soft (bottom, NS283) sorghum grain. PE, pericarp;
SG, starch granule; outside (left) to inside (right) of caryopsis.
T able 1. Categorization of Cultivars, Plant Maturity, Caryopsis Properties, and Amounts of Sormatin and Chitinase in
Sorghums Varying in Mold R esistance
aAntifungal protein (AFP) content and grain mold resistancefrom Buesoet al. (59).bEarly maturity ) <60 days after planting, medium
) 61-80%, late ) >81%.cWhen spreader (S) gene is dominant, tannins accumulate in pericarp and testa.dHard ) <30% abraded
during decortication, medium ) 30-50%, soft ) >51%.eSormatin and chitinase in control caryopses at 30 days after anthesis. Sormatin
and chitinase had coefficients of variation between 11 and 23% for the different cultivars, locations, and age of caryopsis.fMold rating
(1 ) no mold; 5 ) >50% of surface molded) at 50 days after anthesis at College Station, TX.
4734 J. Agric. Food Chem ., Vol. 49, No. 10, 2001Waniska et al.
and pericarp). Type II and III sorghums have dominant
B1 and B2 genes and a pigmented testa (seed coat); type
III sorghums have a dominant spreader (S) gene and
more tannins than do type II sorghums (38).
Phenolic compounds from threemajor categories, that
is, phenolic acids, flavonoids, and tannins, have been
analyzed in sorghum (21, 60). Phenolic acids are deriva-
tives of benzoic or cinnamic acids. Flavonoids consist of
two units: a C6-C3 fragment from cinnamic acid and
a C6 fragment from malonyl-CoA. The major groups of
flavonoids in sorghum are the flavans: flavan-3-en-3-
ols with a double bond between C3 and C4 and the C3
hydroxylated anthocyanidins. Tannins are polymers of
five toseven flavan-3-ol units (catechin) linked through
acid labile carbon-carbon bonds (61-63). Tannin sor-
ghums contain proanthocyanidins as part of their
phenolic compounds but do not contain tannic acid or
hydrolyzable tannins. Phenolic acids and compounds
increaseduring caryopsis development with a maximum
at physiological maturity (and a decrease afterward).
These decreases may be due todecomposition or insolu-
Assabgui et al. (64) reported a good correlation
between thecontent of ferulic acid in maizekernels with
resistance to F. graminearum, whereas ref 65 reports
greater levels of p-coumaric acid in somewhitepericarp,
non-tannin sorghums that were susceptible tomolding.
The conversion of p-coumaric acid to ferulic acid might
be deficient in the susceptible cultivars. Free or bound
phenolic acids in control and inoculated caryopses
correctly classified sorghums into resistant, intermedi-
ate, and susceptiblegroups (Rodriguez-Ballesteros et al.,
Sorghum cultivars resistant to fungal attack con-
tained both a greater variety and larger amounts of free
phenolic acids; this was especially true for tannin
sorghums (62, 65). Red pericarp sorghums are more
resistant tomold than arewhitepericarp sorghums and
contain moreflavon-4-ols (17, 19, 22, 36, 66, 67). A cause
and effect relationship has not been established for
flavan-4-ols nor have their enzyme systems been stud-
No phytoalexin has been detected in the caryopsis of
sorghum. Clive et al. (68) isolated cDNA clones from a
sorghum mesocotyl library after infection with Colle-
totrichum sublineolum that appear toalign partly with
ribonuclease sequences in the database. The level of
chalcone synthase, an enzyme that is involved in the
synthesis of phytoalexin, was greater when mesocotyls
were treated with Cochilobolus heterostophus, which
does not infect sorghum, than with C. sublineolum,
Antifungal Proteins (AF P) in Sorghum Cary-
opsis. Several proteins are constitutively expressed in
developing cereal seeds and have antifungal properties
either in vivo or in vitro. Sorghum grain has not been
studied in as much detail for antifungal proteins as the
more widely cultivated cereals (3, 69-75). However, it
is probable that homologues of many of the proteins,
particularly those present in maize, are alsopresent in
sorghum. In the past few years, several types of bio-
active and antifungal proteins have been identified and
characterized in sorghum, maize, and millet (Table 2).
Sunitha and co-workers (76, 77) identified three
proteins of 18, 26, and 30 kDa, which affected hyphal
growth of F. moniliforme. The 18 kDa component
removes cell wall polysaccharides, whereas the other
proteins areinvolved in leakageof cytoplasmic contents.
The authors concluded that the 18 kDa protein could
be an enzyme acting on cell walls and that the 26 and
30 kDa components could be related to permeatins.
MoreAFP werein hard endosperm, grain mold resistant
sorghums (54, 77).
Seetharaman et al. (78) identified positively charged,
water-soluble proteins belonging tothe permeatin (which
they called sormatin), chitinase, ?-1,3-glucanase, and
RIP groups. Thesynthesis or extractability of antifungal
proteins increased for several AFPs until physiological
maturity and then decreased during desiccation of the
grain (78, 79).
The level of AFP in sorghum caryopis was 7 µg; 6.8-
15 µg was required for inhibitory activity to Fusarium
sp. (76). An AFP fraction containing sormatin, chitinase,
glucanase, and RIP was inhibitory tospore germination
of F. thapsinum, C. lunata, and Aspergillus flavus, all
at 360 ppm (80). Hyphal rupture at the growing tips
was observed for F. thapsinum at 70 ppm and at 70-
360 ppm for C. lunata but not for A. flavus. The amount
of AFPs in sorghum was estimated at 260 ppm in
physiologically maturecaryopses at 30-35 mg/caryopsis
T able 2. Antifungal Proteins of Sorghum, Maize, and Millets
species size/isoform fungal species inhibitedeffective/inhibitory doserefs
sorghum24, 28, and 33 kDa T. viridae
24 kDa, pI 9.8
69, 3, 75
in vitro protein synthesis
in vitro proteinsynthesis
0.7 µg/mL 117, 87, 50
22, pI 9.1
26 and 30 kDa
3, 73, 74
T. reesi, R. solani
sorghum 12, 16, and 20 kDa
mixture of RIP, chitinase,
glucanase, and sormatin
aThe pearl millet protein is a cysteine proteinase inhibitor (118).
SorghumAntifungal Proteins in GrainJ. Agric. Food Chem ., Vol. 49, No. 10, 2001 4735
weight (80), whereas about 70 and 300 ppm was enough
to inhibit F. thapsinum and C. lunata, respectively.
Proteinase Inhibitors. Higher levels of serine pro-
teinase inhibitors were observed in developing hard
versus soft endosperm sorghums (42). Themaizetryspin
inhibitor was implicated for resistance toA. flavus (81,
R ibosome-Inactivating Protein. Type1 RIP barley
grain inhibits the growth in vitro of a number of fungi
(Trichoderma reesi, Botrytis cinerea, and Rhizoctonia
solani), and this activity is enhanced synergistically by
?-1,3-glucanaseor endochitinase(49). Expression of RIP
in transgenic tobacco increased resistance to the soil-
borne fungal pathogen R. solani (83), which was en-
hanced by thecoexpression of barley endochitinase(84).
Transgenic wheat plants expressing thebarley RIP gene
were not protected against Erysiphe graminis (85).
Walsh et al. (86) showed that the maize RIP was
synthesized as a proprotein with a region being removed
during germination. Hey et al. (87) demonstrated that
the connecting hinge between the twodomains of maize
RIP inhibited enzymic activity on ribosomal RNA and
was removed during activation. Sorghum contains
protein bands that reacted with maizeRIP antibody and
had similar molecular weights. Two cross-reacting
protein bands of ∼30 kDa varied in intensity with
sorghum using the same RIP antibody (88). The anti-
body alsodetected the late deposition of the proprotein
in the endosperm during development, and the pro-
protein was split as in maize. The RIP in maize is
located in the aleurone and the scuetellum using tissue
prints and antibodies (89). Seetharaman et al. (78)
observed that the RIP was most extractable from
caryopses at 15 DAA and decreased subsequently.
T hionins/AMPs. A new family of low molecular
weight proteins from sorghum caryopsis that inhibits
R-amylase from insects was identified (90). These
proteins, subsequently called γ-thionins and defensins,
each comprised 47 amino acid residues with four dis-
ulfide bonds (91). A thionin-like peptide fraction identi-
fied as MBP-1 was isolated from maize and was found
to inhibit spore germination and hyphal elongation of
F. moniliforme and F. germinarium in vitro (92).
Chitinase and Glucanase. Several chitinases (three
in the 21-24 kDa range and 28 kDa) and one ?-1,3-
glucanase (30 kDa) were reported in sorghum (93).
Three chitinases were reported in sorghum caryopses
(24, 28, and 33 kDa) that inhibited the growth of
Trichoderma viride and F. thapsinum (94). Maximum
accumulation of chitinase [28 and 33 kDa (78); 27 and
28 kDa (Venkatesha et al., unpublished data)] was
observed at 30 DAA. The caryopsis contained less 27
and 28 kDa chitinase after 24 h of imbibition (during
germination), but theseproteins continued tobepresent
even up to 10 days after imbibition. Several additional
chitinase isoforms [18, 26, and 34 kDa (78); 18.2, 20,
and 24 kDa (Venkatesha et al., unpublished data)]
appeared during germination. Putative clones for chiti-
nase have been derived from a cDNA library of develop-
ing caryopses as well as from genomic DNA. Sequence
analysis revealed very high homology between cDNA
and genomic clones, which, in turn, showed higher
homology with the chitinases a and b of maize (Ven-
katesha et al., unpublished data). An immunoblot study
revealed the polymorphic nature of chitinases in cary-
opses of 24 sorghums, and theamplification and cloning
of chitinase genes from a few of the sorghums are being
investigated (Venkatesh et al., unpublished observa-
Proteins Acting against Aspergillus. The extent
of invasion of the germ in resistant lines might depend
on resistance mechanisms in the endosperm (95). More
?-1,3-glucanase activity was observed in the caryopsis
and callus of maize resistant toA. flavus in response to
infection (96), whereas Chen et al. (81, 82) reported the
presence of higher levels of 14 kDa trypsin inhibitor in
maizeresistant toA. oryzae. Threeunidentified proteins
in sorghum caryopsis inhibited the growth of A. flavus
Twoprotein fractions wereextracted from maizewith
oneshowing growth inhibition of A. flavus and theother
inhibiting aflatoxin formation with littleeffect on fungal
growth (98). Maize protein extracts from resistant
kernels were found to have greater antifungal activity
against A. flavus than did susceptible kernel extracts
in vitro (99).
Variability and R elative Importance of AF P. A
significant inverse correlation coefficient between sor-
matin in caryopses at 30 DAA with sorghum grain mold
rating at harvest time was first observed by Seethara-
man et al. (78) using 32 sorghum culitvars. Extract-
ability of AFPs increased during caryopsis development
and decreased prior to grain drying (77, 78), whereas
deterioration of the caryopsis significantly increases
after physiological maturity.
Sorghum AFP leached from immature caryopses but
was retained in thepericarp of maturecaryopses during
water imbibition (78). If AFPs are mobile and “bound”
to the pericarp during imbibition, then AFPs could be
concentrated in <10% of the caryopsis. This would
increaseAFP concentration ∼10-fold, thereby increasing
their antifungal potential. Moreover, increased quanti-
ties of these proteins were observed after treatment of
flour with protease.
Variations in levels of AFP within the endosperm
were observed, with more AFP being located in the
corneous endosperm of resistant sorghums that contain
more protein bodies (42, 54, 79) and different cell wall
composition (57, 58). Protein bodies and AFPs appear
to be concentrated near cell walls in the corneous
The distribution of AFP within the caryopsis also
varies among sorghums (Waniska et al., unpublished
data). More sormatin was found in the endosperm
(versus pericarp or embryo) of Dorado (resistant, hard
endosperm sorghum) and in the pericarp (versus en-
dosperm or embryo) of TX2536 (susceptible, intermedi-
ate-endosperm-texture sorghum). More chitinase was
found in the endosperm (versus pericarp or embryo),
whereas more?-1,3-glucanasewas found in thepericarp
(versus endosperm or embryo). Soaking decreased the
extractability of both chitinase and glucanase from
physiologically mature caryopses.
Induction of Antifungal Proteins. Sormatin-like
proteins were induced after fungal infection (77). Bueso
et al. (59) determined that sormatin was induced by
infection in vivo in resistant sorghums when the sor-
ghum caryopses were stressed with grain mold fungi
at anthesis and sampled at physiological maturity. Also,
periodic sprinkling of panicles in the field, similar toin
vitro imbibition, decreased levels (or extractability) of
AFP in susceptible sorghums at physiological maturity.
4736 J. Agric. Food Chem ., Vol. 49, No. 10, 2001Waniska et al.
Rodriguez-Herrera et al. (43) compared the levels of
four AFPs using eight mold resistant and eight suscep-
tible lines derived from a susceptible by resistant cross,
grown in eight environments over three years. Infection
with grain mold resulted in the induction and/or reten-
tion of more AFPs in the resistant lines (Figure 2),
suggesting that the coexpression of four AFPs may be
required to confer resistance in lines with a nonpig-
mented testa. The ?-1,3-glucanase levels in resistant
lines, however, did not increase as much as other AFPs.
Chitinase, sormatin, and RIP concentrations were 1.5-
14-fold higher in the resistant lines compared to sus-
ceptible lines and were associated with grain mold
Puncturing the germ induced chitinase synthesis in
mature Dorado caryopsis but not in TX2536 caryopses
(Waniska et al., unpublished observations). Puncturing
and soaking induced sormatin mobility and accumula-
tion in caryopses tissues of Dorado and TX2536.
Infection of maize seeds with F. moniliformeresulted
in induction and accumulation of antifungal proteins,
and such response was found to be more rapid in
germinating embryos (100). Antifungal activity of maize
proteins upon imbibition and germination was at-
tributed to fractions that reacted to zeamatin and RIP
Genetic Markers Associated with Grain Mold.
Studies of other cereals demonstrate that it is possible
toidentify biochemical or molecular markers, which can
be exploited to follow resistance in breeding programs.
Mingeot and J acquemin (102) found high polymorphism
for one marker, which was subsequently shown to
encode a thaumatin-like protein, observing many pat-
terns in ∼48 cultivars of wheat analyzed. Farris et al.
(103) reported the use of 508 genetic markers including
a large number of candidate genes in screening a
population of 114 recombinant inbred lines between a
hard red spring wheat and a synthetic hexaploid wheat
(derived from Triticum turgidum and Aegilops tauschii).
Theoxalateoxidasegenewas a good marker for tan spot
resistance using a pathotype avirulent to Lr23. A per-
oxidase gene was linked toboth resistance and Lr23. A
phenylalanine ammonium lyase gene and a thaumatin
gene appeared tobe linked tothe resistance genes Lr27
and Lr31. The disease resistance genes appeared clus-
tered on the 7BL chromosome. Markers encoding chal-
cone synthase and a chitinase were associated with
karnal bunt resistance. Ittu et al. (104) showed that
resistance toFusarium head blight was associated with
thegliadin loci, Gli-B1 and Gli-D1, which is reminiscent
of the positive relationship between prolamins and
resistance to grain mold in sorghum. de la Pena et al.
(105) similarly found QTL associated with resistance to
Fusarium head blight in barley. The disease-resistance
genes (R-genes) map toareas very close toknown genes
for resistance (106, 107). Mutations in resistance genes
may also account for differences between susceptible
and resistant varieties. Klein et al. (108) have identified
five QTLs associated with grain mold. Work is under-
way todetermine the location of genes for AFP content
in sorghum and their location relative tomapped grain
mold resistance QTLs (108).
R esistance Genes. Resistance (R) genes present in
the host plant may be activated on infection by patho-
genic fungi, leading via signal transduction pathways
to defense responses, such as the production of phyto-
alexins and antifungal proteins (discussed above). The
relationship between the R genes and pathogens may
be extremely specific, resulting in responses only to
specific species or races of pathogens. We have shown
above that sorghum grain protects itself in a very
elaborate manner using several mechanisms. The syn-
thesis of the endoplasmic reticulum, the prolamins, the
antifungal proteins, and the cell wall may be coordi-
nated by the R genes.
In recent years R genes have been cloned from a
number of plant species, using either mapping ap-
proaches (“map-based” cloning) or transposable ele-
ments tofacilitategeneidentification and isolation. This
has shown that R proteins sharecommon features, most
notably the presence of leucine-rich repeats which may
be combined with protein kinase domains. Moreover,
resistance genes appear to be clustered in maps (109,
110). This has facilitated the isolation of further R
genes, using easier PCR-based strategies.
R genes have not so far been isolated from sorghum.
However, their isolation by PCR-based technology should
be facilitated by the availability of R gene sequences
from other species including lettuce (111), tomato(112),
soy (107, 110), rice (113), potato(114), and maize (106).
Both conserved and variable sequences are observed
between different R genes, and theformer could beused
F igure 2. Antifungal protein levels (micrograms per gram)
in mature caryopses of eight grain mold resistant (GMR) and
eight grain mold susceptible (GMS) lines grown at seven
environments. Resistant lines havemoreAFP than susceptible
lines except for glucanase. For glucanase, means followed by
the same letter are not significantly different (within environ-
SorghumAntifungal Proteins in GrainJ. Agric. Food Chem ., Vol. 49, No. 10, 2001 4737
todesign PCR primers or oligonucleotides toisolatepart
or whole of the disease resistance genes from sorghum
(115). Oncethegenes or fragments thereof areavailable,
their relationship with resistance to grain mold could
be investigated by assaying for the expression of these
genes in developing seed and their linkage with resist-
ance in a breeding program.
Studies of other systems have shown that overexpres-
sion of R genes may lead to broad-spectrum resistance
with high levels of antifungal proteins and other defense-
related compounds (116). Transgenic plants have been
made with both the antifungal proteins and the kinases
and leucine-rich repeat proteins that are involved in
signal reception and transduction. Transgenic plants
made with specific antifungal proteins may be able to
resist certain fungi, whereas a broad-spectrum resist-
ance may occur when kinases are used. Overexpression
of such genes in the tissues of developing panicle of
sorghum at the time of susceptibility to grain mold
infection could lead to increased resistance.
Prospects for the F uture. Transformation of crop
plants is a promising and powerful tool toprotect them
from biotic and abiotic stresses and will be of immense
value in preventing yield losses, which is an important
component of yield stabilization in areas where these
stresses are endemic. Even though sorghum is a hardy
plant capableof withstanding biotic and abiotic stresses
to some extent, further increases in resistance to
stresses could result in yield enhancement. This strat-
egy may be important for dealing with the ever-
increasing world requirement for food and feed. In the
future, other agronomically useful genes, such as the
genes involved in the biosynthesis of cellulose and
lignin, which strengthens the stalks, may be utilized to
prevent lodging. Additionally, genes that confer resist-
ance to insects could be introduced into sorghum to
reduce yield loss from insect feeding and to reduce
opportunistic fungal infections at the wounding sites.
Genes for phytase can reduce the phytic acid content of
plants toincreasetheir nutritional valuetoanimals and
to reduce phosphate pollution due to excretion of
phosphates by livestock. Additionally, transgenic sor-
ghum plants could be a source of pharmaceuticals and
biopolymers and thus serve toincrease the commercial
value of the sorghum crop. We have started a long-term
project tointroducethesegenes in various combinations
intosorghum inbreds in order toimprove the resistance
to stalk rot.
CONCLUSIONS AND RECOMMENDATIONS
Current sources of resistance provide only partial
protection against stalk rot and grain mold fungi, the
most important of which are Fusarium, Alternaria, and
Curvularia. Grain mold resistance can derive from a
combination of characteristics (pigmented testa, phe-
nolic compounds, red pericarp, endosperm texture, cell
wall, and AFP), which act additively or synergistically.
Different resistance mechanisms could be linked by the
R genes. Some of the sources of resistance have a
negative impact on end use properties. Hence, there
may be a tradeoff between protection and quality.
Pigmented Testa and R ed Pericarp. Becauseboth
red pericarp and tannins are related to grain mold
resistance, utilization of red pericarp sorghums (grown
on a tan plant) and tannin sorghums are reasonable
short-term solutions to decrease grain molding. Deter-
mination of the causative agents in these sorghums, for
example, nonpigmented phenols, other compounds, and
tannins, against fungi is needed. Then, the enzymes,
their regulation, and their molecular biology need tobe
Grain Hardness. Hardness corresponds to high
levels of γ-kafirin. Thebiochemical basis for this effector
needs to be understood and the existence of modifier
genes (as in QPM) established. The effect of introducing
more gamma genes into sorghum on mold resistance
needs to be studied. In addition, hardness is currently
associated with high levels of antifungal proteins, and
this linkage needs to be understood and then broken.
Antifungal Proteins. A link between AFP and
resistance is clearly established in both stalk rot and
grain molding, although only partial protection is
obtained. In the short term it will be possible to select
for high levels of characterized AFP (sormatin, RIP,
?-1,3-glucanase, and endochitinase) in developing, ma-
ture, and infected grain by direct analysis or by using
antibodies or molecular markers. More information is
required on the endogenous sorghum AFP to identify
new components, determine activity and synergism,
determine location and amounts in developing and
mature tissues, determine initiation of synthesis, and
relate the above to resistance.
Marker-Assisted Selection. The development of
molecular markers for the sorghum genome (e.g., SSRs)
is essential to underpin the selection of resistance and
the combination of resistance with yield and end use
quality. It will also facilitate dissection of the various
aspects of resistance, for example, the separate effects
of hardness and AFPs. A more direct approach could
also be adopted using markers based on characterized
antifungal proteins (e.g., thaumatin) and putative R
genes (based on the other species).
T ransformation. Transformation is an essential
prerequisite for long-term improvement and must con-
tinue to be supported. Transformation should initially
express sormatin, ?-1,3-glucanase, and endochitinase
under a strong endosperm specific promoter. Further
transformation may requirespecific promoters tocontrol
the level and tissue specificity of expression, for ex-
ample, glume, developing endosperm, or ovary wall/
Wearevery grateful for thehard work and dedication
of legions of former graduate students and colleagues
who have provided so much inspiration and reliable
information for our programs. The long-term support
of the Texas and Kansas Agricultural Experiment
Stations, privateindustry, INTSORMIL, and CFTRI are
appreciated. The permission accorded by the Director
of CFTRI topresent this paper is acknowledged by A.C.
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Received for review J anuary 2, 2001. Revised manuscript
received August 9, 2001. Accepted August 10, 2001.
4742 J. Agric. Food Chem ., Vol. 49, No. 10, 2001Waniska et al.