DANIEL McALPINE MEMORIAL LECTURE
Biology
and management of Phytophthora spp. attacking field crops in Australia
Presented
at the Eleventh Conference of the Australasian Plant Pathology Society, Perth,
Western Australia, 29 September – 2 October 1997.
Introduction
The
many and varied achievements of Daniel McAlpine, the ‘Father of Australian
plant pathology’ have provided the members of the Australasian Plant Pathology
Society with a proud legacy. Although McAlpine is best known for his work
while employed as Vegetable Pathologist by the Victorian Department of
Agriculture, he had already established a distinguished research career in Scotland
before migrating to Australia. His early days in Australia were spent teaching
at Ormand College, Melbourne, where he also performed with distinction. McAlpine
is probably best known for his work with rusts and by 1906, he had described 75
new species. His interests were wide-ranging, however, and have been summarised
by Fish (1976).
This memorial lecture concerns the biology and
management of Phytophthora spp. attacking field crops.
Phytophthora
is one of the world’s most economically significant plant pathogens. The
etymology of Phytophthora is ‘plant destroyer’, with the best known
member of the genus being its type species, Phytophthora infestans, the
cause of potato late blight. It is sobering to reflect that although more
than 150 years have now elapsed since the devastating late blight epidemics
first gripped Ireland and other parts of Europe and North America, P.
infestans is today still causing substantial economic losses in these same
regions. This clearly indicates that the genus Phytophthora provides an
apparently inexhaustible supply of challenges to plant pathologists, and we are
a long way from sufficiently understanding its biology to effect sustainable
disease management. The impact of Phytophthora worldwide has been
addressed recently by Erwin and Ribiero (1996), in their excellent and
comprehensive monograph on the genus. Phytophthora causes serious
diseases on all of the major plant groups, with the exception of the monocots.
In the United States of America, disease costs (control and production losses)
are estimated at billions of dollars annually (Erwin and Ribiero 1996), and in
Australia at greater than $200 million annually (Cahill 1993; Irwin et al.
1995). Also, in Australia Phytophthora has caused major epidemics in
natural vegetation (Podger 1972) and the costs of these to society are hard to
estimate.
In this lecture I will concentrate on the biology and
control of Phytophthora-incited diseases of field crops in Australia, an
area where I have maintained a research interest for the last 25 years. This
discussion will consider the origins and phylogenies of the major Phytophthora
spp. attacking field crops in Australia (Table 1), with additional emphasis on
the biology of the host-parasite interactions. This will be approached through
consideration of a small number of case histories, each exploring improved
management through an increased understanding of the genetics of the
host/parasite interaction.
It
is now clearly established that the Oomycetes, of which Phytophthora is a
member, belong in the kingdom Chromista (Dick 1995a and b) and as
such have evolved from heterokont photosynthetic brown algae, which lost their
chloroplasts. Evidence for this includes: heterokont flagellation of zoospores,
cell walls predominantly cellulose, tubular cristae in the mitochondria and
diploid somatic phase (Gunderson et al. 1987). These characters, as well
as several others, clearly distinguish the Oomycetes from the ‘true’ fungi
such as Asco-mycetes and Basidiomycetes, and for this reason they are placed in
the kingdom Chromista.
Traditional
taxonomy of Phytophthora places a lot of emphasis on the sporangial
morphology, in particular whether sporangia are papillate, semi-papillate or
non-papillate (Waterhouse 1956). The second most important character is
sexuality, combined with antheridial type (amphigynous and/or paragynous). A
classification based on these characters gives rise to six groups (Waterhouse
1956), and this system forms the basis of the most recent classification system
for Phytophthora (Stamps et al. 1990).
While this system has proven useful to plant
pathologists over the last few decades, it has become obvious that several of
the morphological species are polyphyletic assemblages e.g. P. megasperma,
comprising a number of genetically distinct and reproductively isolated groups
(biological species) with widely differing host ranges. Thus, the challenge is
to use criteria other than morphology which will provide a more natural
classification for Phytophthora, and which will be more generally useful
to plant pathologists.
The
genes encoding ribosomal RNA (rRNA), because they comprise conserved and diverse
regions, have been found to be informative at many taxonomic levels (Hibbet
1992). The coding regions (18S, 5.8S and 28S rRNA) are highly conserved, and
have application at the suprageneric level and higher, whereas the internal
transcribed spacer regions (ITS1 and ITS2), which lie between the 18S and 28S
genes, are more variable, and the intergenic spacer region (IGS, between each
rRNA tandem repeat) is even more variable (Figure
1).
Over the last few years in the Cooperative Research
Centre for Tropical Plant Pathology, we have embarked on a program to gain an
increased understanding of Phytophthora phylogenies through: use of
molecular markers (RAPDs, RFLPs) to study diversity levels within morphological
species and to identify representative isolates; application of nucleotide
sequencing of the ITS and IGS regions of the rRNA to the representative isolates
as deduced from the molecular marker studies; and construction of phylogenies
based on nucleotide sequence homology. In some of the earlier studies, using
protein profiles (Irwin and Dale 1982), it was apparent that P. megasperma
contained at least two biological species, one host specific to soybean, now
called P. sojae, and the other with specificity towards lucerne and
chickpea, and now designated P. medicaginis. Within the morphological species
P. megasperma there are at least two other biological species (as yet
unnamed) which attack chickpea (Liew and Irwin 1994). These findings have
subsequently been confirmed through fragment analysis (Whisson et al.
1993), and rRNA nucleotide sequence analysis (Crawford et al. 1996). A
phylogram based on ITS1 and ITS2 regions confirmed the polyphyletic structure of
the morphological species P. megasperma, with its former members
appearing in different clusters (Figure 2).
Several other questions are raised by the groupings in
Figure 2. Have host specific homothallic biological species such as P.
sojae and P. vignae evolved from the heterothallic and broad host
range species P. cinnamomi? A similar question can be asked for P.
medicaginis and P. trifolii, two homo-thallic biological species
which are related to members of the heterothallic P. cryptogea/P.
drechsleri complex. From an evolutionary perspective, sexuality and
antheridial attachment type would seem to be less important than sporangial
papillation, which broadly splits Phytophthora into two main groups,
non-papillate and papillate/semi-papillate. Gäumann and Wynd (1952) proposed an
evolutionary advance in the Peronosporales from soilborne unspecialised Pythium
spp., through Phytophthora to the downy mildews. It could be argued
that within Phytophthora, there has been an evolutionary advance from
the soilborne, non-papillate species to the papillate and largely airborne
species, which show the closest affinity to the downy mildews. In the papillate Phytophthora
species, sporangia are often deciduous and airborne, and in the downy mildews
we see an even further advance to where, in some genera e.g. Peronospora
and Perono-sclerospora, sporangia always function as conidia, without
zoospore release (Shaw 1981).
Within
both the papillate and non-papillate Phytophthora spp., we see an almost
complete range of pathogenesis expressed, from apparent necrotrophy and a broad
host range (P. cinnamomi and P. parasitica) through to
hemibiotrophy and the production of rudimentary haustoria in host cells (P.
sojae and P. infestans). With the latter there is specialisation
towards host species and cultivars, while with the former, this specialisation
is lacking. For P. cinnamomi, the issue of necrotrophy is also not
unequivocal. Davison et al. (1994) showed that after wound inoculation of
woody stems and roots of jarrah and pine, the fungus could be isolated from
symptomless phloem well in advance of the necrotic lesion. It can be argued that
the species with broad host ranges have developed mechanisms which avoid
provoking effective host defence responses. However, as hemibiotrophy involves
keeping the invaded plant cell alive while avoiding host defence responses for
the first 24 h or so of the interaction, this would indicate highly evolved
specific adaptations on the part of the pathogen. This is further indicated by
the extensive intracellular development of the hemibiotrophs, compared to the
necrotrophs which grow intercellularly, and largely invade killed cells. Species
such as P. sojae and P. vignae may have evolved from a
heterothallic common ancestor similar to P. cinnamomi (Irwin et al.
1997). If a mutation occurred in one of the mating types, allowing it to attack
soybean or cowpea, and sexual reproduction involving the mutant could only occur
in the new host, then the mutant would be under strong selection for asexual
reproduction and selfing, possibly ultimately leading to homothallism and
genetic separation from the original heterothallic population. Sympatric
speciation events currently seem to be a likely source of the homothallic and
host specific Phytophthora species.
Recently, there has been a lot of interest in a group
of proteins found almost universally in Phytophthora, the elicitins
(Grant et al. 1996). It is not known whether the role of these compounds
is as avirulence determinants or pathogenicity factors. Nucleotide sequencing of
genes encoding elicitins from a range of Phytophthora species may provide
insights into evolutionary trends and possible roles for these molecules.
P.
medicaginis/lucerne and
chickpea Application
of genome-scanning, molecular markers such as RAPDs to isolates of P.
medicaginis collected in both Australia and the United States of America has
demonstrated that this biological species shows low levels of diversity at the
DNA level. This could indicate that it is a relatively recent immigrant to both
countries; perhaps greater diversity levels exist in the centres of diversity of
the hosts, which for Medicago and Cicer includes Transcaucasia.
While this disease in lucerne can be readily identified based on symptomatology,
Liew et al. (1997) have designed PCR primers that will amplify from the
IGS a 364 bp fragment specific for P. medicaginis. These primers will
detect template DNA as low as 4 ng and in a host pathogen ratio of 1 000 000 :
1, and have been used for detection of P. medicaginis in field infected
roots. The success with this disease provides a model for development of
DNA-based detection methods for other Phytophthora pathogens,
particularly those of most concern to the Nursery Industry. Detection directly
from soil samples still presents an unresolved challenge.
Cultivars of lucerne resistant to Phytophthora root rot
have now been deployed in Australia and North America for almost 20 years.
Interestingly, up to now, pathogenic specialisation has not been an issue; the
reasons for this are worth exploring.
Cultivated lucerne is both autotetraploid (2n = 4x =
32) and outbreeding, both of which preserve heterogeneity. Because lucerne shows
high levels of inbreeding depression, cultivars are developed as synthetics with
a wide genetic base, and thus heterogeneity within a cultivar is preserved.
Several different genetic systems conditioning resistance to P. medicaginis
in lucerne have been identified (Irwin et al. 1995). An example comes
from the detailed studies of a 4x clone, designated M193, which at 4x level
appeared to have Phytophthora resistance conditioned by two complementary
dominant genes, Pm1
and Pm2 (Irwin et al. 1981). When this same clone was scaled
down to the diploid level, by interploid bridge crosses, inheritance studies
revealed two different modes of inheritance in the four different derived
diploid plants. In two, resistance was inherited as a dominant allele at both of
two independently segregating complementary loci, Pm1
and Pm2
(as at 4x level), and in the other two, resistance was inherited as a dominant
allele at either of two independently segregating loci Pm5
and Pm6 (Havey et al. 1987). Thus, the single plant M193 appears
to contain at least four independent loci conditioning Phytophthora
resistance. S1
plants from this clone have been used in Australia in a breeding program to
develop a commercial cultivar with the highest levels of Phytophthora
resistance known worldwide (Waterhouse and Williams 1993).
To
further exploit variability for resistance to P. medicaginis existing at
the diploid level, the derived diploids of M193 were crossed with diploid clones
of M. falcata containing two independent dominant genes Pm3
and Pm4.
These diploid F1s were then used as females, and crossed with a tetraploid
anthracnose resistant clone as the male. From 0.5–24.2 tetraploid progeny were
obtained for each 100 diploid flowers pollinated, through unreduced female
gametes. These 4x progeny were polycrossed to provide a tetraploid population
(WAPRS-4) containing genes Pm1
through to Pm6 plus An1
for anthracnose resistance (Havey and Maxwell 1987; Havey et al. 1989).
This germplasm has been particularly useful in transferring resistance to P.
medicaginis in several breeding programs.
Thus, it can be argued that the population buffering
afforded by the simultaneous use of different clones with different genetic
systems conditioning resistance, in combination with autotetraploidy and
outbreeding which both preserve heterogeneity, have provided a stable
agro-ecosystem, which is in marked contrast to the rapid evolution of new Phytophthora
races which has occurred following deployment of pure lines of soybean and
chickpea.
The situation is different for chickpea; the species
generally shows less resistance to P. medicaginis, even after extensive
screening of world chickpea germplasm collections (Brinsmead et al.
1985). From this, we can postulate that the interaction between P.
medicaginis and chickpea is a relatively new encounter, in contrast to
lucerne. Both hosts have centres of diversity (but for chickpea not necessarily
a centre of origin) in overlapping regions of Transcaucasia and searches of
those regions for the pathogen could provide valuable collections which could be
used to further address some of these unanswered questions.
P.
vignae/cowpea This pathogen and disease were first described by Purss
(1957) in Queensland. Since then, P. vignae has been reported from West
Africa, Sri Lanka and Japan (Erwin and Ribiero 1996). There are two strains of P.
vignae, the Australian strain which attacks cowpea and adzuki bean, and a
strain from Japan which is pathogenic on adzuki bean but not pathogenic to
cowpea (Kitazawa et al. 1978; Kitazawa et al. 1979; Liew et al.
1991). There is clear-cut pathogenic specialisation within cowpea-attacking
strains of P. vignae (Purss 1972), suggesting a long history of
co-evolution with its Vigna hosts. Diversity studies made on the two
pathogen groups using RFLPs showed a high level of overall similarity (F, the
percentage of bands in common = 94%) between the Australian and Japanese
isolates, with low molecular diversity (F > 97%) within the Japanese and
Australian isolates. These results with relatively small numbers of isolates
strongly suggest that P. vignae has not evolved in Australia, but most
likely has a centre of diversity in Indo-China along with cowpea. We can also
conclude that the cowpea and adzuki bean strains have recently shared a common
ancestor; had they arisen independently in Japan and Australia, they would not
be expected to share so much overall homology.
Although
cowpea is only a minor crop in Australia, cultivars exhibiting monogenic and
race-specific resistance have been quickly overcome by new races of P.
vignae (Purss 1972). Partial resistance has been known to exist in cowpea to
P. vignae since the 1950s (Purss 1972), but it has not been utilised.
Davis et al. (1994) characterised expression of partial resistance under
field and glasshouse conditions by measuring disease incidence, disease
severity, time lag before onset of disease, relative area under the disease
progress curve and apparent mortality and infection rates on a range of lines
which showed susceptibility following hypocotyl inoculation (used to assay for
complete or race-specific resistance). The character found to be most useful in
differentiation of relative levels of partial resistance was final percentage
of plants with non-killing lesions. One line, Blackeye-5, which manifests
partial resistance in the field to all races of P. vignae, shows complete
susceptibility following hypocotyl inoculation with race 3 (Purss 1972). The
expression of the partial resistance in cowpea, including Blackeye-5 to race
3, can be assessed in seedlings provided inoculum levels are strictly controlled
(Davis et al. 1993), and exposure to inoculum is through a natural
infection court by sowing seeds into infested soil. The partial resistance
expression in Blackeye-5 does appear to have a race-specific component, since it
can be overcome by race 4 in the glasshouse at inoculum levels which still allow
expression of resistance by other partially resistant lines (Davis et al.
1993). Interestingly, the single gene in Blackeye-5 which confers complete
resistance to race 1, when transferred to Poona in a backcrossing program to
create Caloona, was completely ineffective in the field against race 3. This
indicates that the background genotype of Blackeye-5 is important in conferring
partial resistance. We have used Blackeye-5 in a pedigree program as a parent in
a cross with Bechuana White (resistant to races 1 and 3), and selected for both
complete and partial resistance in the F3 lines and subsequent generations, to generate Ebony PR, which
expresses complete resistance to some races and partial resistance to all
remaining known races of P. vignae.
P.
sojae/soybean The level of understanding of the genetics of the P.
sojae/soybean interaction is much further advanced than for P. vignae.
However, there are a lot of similarities between the two interactions which is
not surprising given the close genetic relationship between P. sojae
and P. vignae. For P. sojae and soybean, a clear gene-for-gene
relationship has been established. In the host, 13 different dominant resistance
genes at seven loci have been characterised, while in the pathogen avirulence is
dominant and generally appears to be monogenic (Whisson et al. 1995).
Interestingly, close linkage was observed between Avr 4 and Avr 6 (0.0 cM), Avr
1b and Avr 1k (0.0 cM) and Avr 3a and Avr 5 (4.6 cM).
Diversity studies utilising DNA markers show that the
Australian populations of P. sojae are genetically uniform, compared to
their United States counterparts. This strongly suggests that P. sojae is
a relatively recent immigrant to Australia, and that the five races that have
been identified in this country have evolved from a common genetic background.
An analysis of resistance genes deployed in Australia and the occurrence of new
races clearly shows a correlation between the deployment of a particular gene(s)
and the evolution of a matching race (Drenth et al. 1996).
Where there is a strong gene-for-gene relationship as
described above, questions concerning the origins of the interacting genes and
their previous and present functions are invoked. Until the genes are cloned and
their functions determined, one can only speculate about their role. Using the
model proposed by Keen (1982), basic compatibility must first be established
between the host and parasite, and a gene-for-gene relationship is then
presumably superimposed over this. However, host specificity is not complete for
P. sojae, which is also known to attack lupin (Jones and Johnson 1969).
The functional relationships between genes conditioning host and cultivar
specificity are interesting to contemplate. For example, in the situation with P.
sojae, we could presume that the same genes condition basic compatibility to
both lupin and soybean, but these are functionally different to those involved
in cultivar specificity. It could be that P. sojae has had a long
co-evolution with soybean in the centre of diversity in Indo-China, and only
recently encountered lupin after movement into the United States of America.
Recent work by Lohnes et al. (1996) has shown that the highest incidence
of soybean lines resistant to P. sojae occurs in the eastern Chinese
provinces of Anhui and Jiangsu, suggesting that this has been the centre of
co-evolution. The interaction between P. medicaginis and chickpea may
also be a new encounter, since resistance does not appear to exist in chickpea,
yet is extensive in Medicago. In bacterial plant pathogens, which
represent considerable less complexity, the hrp gene clusters and the avirulence
determinants are apparently separate entities. Without heterologous probes, to
clone genes conditioning host species specificity by positional cloning requires
interspecific crosses. This may not be completely out of the question for P.
sojae and P. vignae, which share a relatively close homology at the
DNA level. Given their relatively close affinity to P. cinnamomi, will
functionally related genes to those determining host and cultivar specificity
in P. vignae be found in P. cinnamomi?
Over
the last decade, we have seen substantial increases in our understanding of
the biology of host/parasite interactions through the application of techniques
in molecular biology, particularly selection neutral DNA markers. As a research
tool, molecular technology has already proven itself. At a practical
application level, however, the benefits are yet to emerge in agriculture. The
first applications will be in DNA-based identification, and we are already
seeing these technologies being applied in the development of quarantine
strategies. If they are to be used in routine disease diagnosis, then probably Phytophthora
represents one of the most important potential applications. Technical issues
such as DNA extraction from soil, and licensing agreements for already existing
patents over the use of PCR will have to be resolved before it will be
technically and economically possible to implement wide application.
Novel resistance genes also have the potential for
significant impact in the improved management of Phytophthora. We are now
testing novel constructs in a variety of situations, including constructs
encoding direct antifungal activity or transgenic expression of host defence
genes, e.g. peroxidases. These approaches should be viewed as complementary to
conventional resistance breeding, and they are probably going to have the
greatest impact once modes of action and pathogenesis mechanisms are determined.
The disease caused by P. medi-caginis in chickpea, where no naturally
occurring resistance expressed at high levels is available in chickpea,
represents an obvious application system for this technology. In important
agricultural and horticultural commodities, this approach also has application
for control of diseases caused by P. cinnamomi, where naturally occurring
resistance in the hosts of interest is hard to find.
I believe the greatest advances in plant disease
management are going to eventuate once the molecular and functional basis of
host and cultivar specificity are understood. Even the work just described is
largely empirical, and without an understanding of mechanisms, probably unlikely
to lead to sustainable disease management. There is an ongoing need for basic
research into these processes, and the P. sojae/soybean interaction,
because of the extensive research which has already been done at the genetic and
biochemical levels in several laboratories, presents an ideal system for the
study of fundamental host/pathogen recognition processes. In the meantime,
conventional plant breeding can be made far more efficient through the
application of DNA-marker assisted selection, a development of DNA technology
which now has wide acceptance and is fast becoming routinely used.
The
work reported here represents interactions with a large number of colleagues
over a 25 year period. In particular, Mr. G.S. Purss contributed substantially
at the outset of my career to the work discussed. His, and the contributions
of many others, are gratefully acknowledged.
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Figure 1 Diagrammatic representation of the organisation of the ribosomal DNA repeat unit in Phytophthora.
Figure 2 Single most parsimonious phylogram generated from branch and bound algorithm in Paup 3.1.1. The percentages are the frequencies with which a given branch appeared in 500 bootstrap replications. P, SP and NP represent the sporangial classes of papillate, semipapillate and non-papillate, respectively. NP* indicates non-papillate, previously classified as semipapillate. A–D indicate the four main branches. Horizontal separation between nodes of the trees is proportional to phylogenetic distance. The tree was rooted by making Achlya bisexualis the outgroup. (Reprinted with permission from Crawford et al. 1996.)