Diversity, Pathogenicity, and Management of Verticillium Species

TL;DR: This review focuses on Verticillium dahliae, placing emphasis on the controversy surrounding the elevation of a long-spored variant as a new species, recent advances in the analysis of compatible and incompatible interactions, highlighted by the use of strains expressing fluorescent proteins, and the genetic diversity among Verticills spp.

Abstract: The genus Verticillium encompasses phytopathogenic species that cause vascular wilts of plants. In this review, we focus on Verticillium dahliae, placing emphasis on the controversy surrounding the elevation of a long-spored variant as a new species, recent advances in the analysis of compatible and incompatible interactions, highlighted by the use of strains expressing fluorescent proteins, and the genetic diversity among Verticillium spp. A synthesis of the approaches to explore genetic diversity, gene flow, and the potential for cryptic recombination is provided. Control of Verticillium wilt has relied on a panoply of chemical and nonchemical strategies, but is beset with environmental or site-specific efficacy problems. Host resistance remains the most logical choice, but is unavailable in most crops. The genetic basis of resistance to Verticillium wilt is unknown in most crops, as are the subcellular signaling mechanisms associated with Ve-mediated, race-specific resistance. Increased understanding in each of these areas promises to facilitate management of Verticillium wilts across a broad range of crops.

Summary

INTRODUCTION

• The genus Verticillium encompasses a cosmopolitan group of ascomycete fungi, including several phytopathogenic species that cause vascular wilts of plants.
• When this type of resistance is compromised, Verticillium wilt is likely to re-emerge as a significant production problem for such crops.
• All other strains of V. albo-atrum are referred to as Grp1 (13).

CONTEMPORARY TAXONOMIC CONTROVERSY

• A variant strain of V. dahliae from horseradish was described in 1961 by Stark (13).
• If the long-spored strains are the ancestral form, this may explain their relatively narrower host range compared with V. dahliae (17, 149) and potentially answer questions raised by Clewes & Barbara (25) regarding the nature of the host where the hybridization may have occurred.
• Hybridizations among potential progenitors could occur repeatedly in multiple crucifers or other hosts, and could include multiple Verticillium spp. or other fungal parents.
• Furthermore, the observed DNA polymorphisms were not consistently associated with either Verticillium species, in some cases the long-spored strains shared SNPs with V. dahliae, whereas in others they shared them with V. albo-atrum.

A NOVEL LOOK AT THE VERTICILLIUM DISEASE CYCLE

• Recent advances in fungal transformation techniques and the transgenic expression of fluorescent proteins, like the green fluorescent protein (GFP) from Aequoria victoria, have enabled an unprecedented view of plant-fungal interactions, especially when combined with the use of confocal microscopy (101a).
• Most studies of V. dahliae using immunoenzymatic and histological staining techniques observed colonization of the root cap and within the zone of root elongation on cotton (58), potato (20, 112), and sainfoin (68).
• En masse, these findings suggest that successful vascular infection of the host is probably more dependent on the initial colonization site, rather than the frequency of root colonization.
• Successful vascular infections at the root cap on lettuce were associated with sparse growth of hyphae directly towards vascular tissues with little hindrance, whereas infections originating from the zone of root elongation developed more extensive inter- and intracellular colonies before invading vascular tissues.

OF WEED HOSTS

• Seed transmission of V. dahliae has been documented in numerous crops in addition to The Verticillium wilt disease cycle on lettuce as observed using a GFP-tagged strain of V. dahliae.
• (b) Tip of a lateral root colonized by V. dahliae with simple conidiophores protruding from root surface, 12 days after inoculation. (c) A longitudinally dissected lettuce root exhibiting advanced colonization of cortical and vascular tissues, 2 weeks after inoculation.
• Of equal concern is the potential of seed transmission among weed hosts of Verticillium spp.

POPULATION BIOLOGY

• The population biology of V. dahliae has been primarily addressed on the basis of VCGs (92) and several molecular markers including RAPDs (17, 87), restriction fragment length polymorphisms (21, 103, 104), amplified fragment length polymorphisms (27, 29), and specific primers (110).
• Strains that showed a particular molecular haplotype were distributed over different geographic locations in both countries (88).
• Collins et al. (29) found that groups α and β of the long-spored Verticillium strains from crucifers formed two clusters showing more than 60% similarity.

MANAGEMENT OF

• Because of its inaccessibility during infection, long-term persistence in the field, broad host range, and scarcity of resistance in host germplasm, control of Verticillium wilt has relied heavily on soil fumigation (44, 109), but is contingent on the economic returns from the crop.
• Because of the sitespecific nature of soil pH and organic matter, efficacy of these products in pathogen suppression is inconsistent.
• Genetic analyses of Verticillium wilt resistance in tomato were determined by Schaible et al. (131), demonstrating that a single dominant allele conferred wilt resistance against V. albo-atrum and was designated as Ve.
• Unlike many other R proteins, both Ve proteins possess a cytoplasmic C-terminus similar to sequences that stimulate receptormediated endocytosis in mammalian cell surface receptors (80).

CONCLUDING REMARKS

• Verticillium dahliae and V. albo-atrum are phytopathogenic species that differ markedly in host range and the types of survival structures produced.
• Because the taxonomy of Verticillium species has implications for the systematic study of pathogenic strains and their management, this is an area of research that deserves additional attention.
• The newly available genome sequences of V. dahliae, V. albo-atrum (85, 164), and the limited set of sequences of long-spored crucifer strains have empowered studies to address some of the topics discussed in this review through comparative genomics.
• Increased understanding of these relationships is likely to offer novel strategies to manage soilborne diseases, including avoidance.

ACKNOWLEDGMENTS

• The authors are grateful for the presubmission reviews from Dez Barbara, Steve Goodwin, Ryan J. Hayes, Beiquann Mou, and Ivan Simko.
• The authors thank their collaborators of the grant entitled “Verticillium comparative genomics-understanding pathogenicity and diversity,” USDACSREES-NRI proposal number 0627011.
• Furthermore, funding from the USDA-CSREES-CAR program, the California Leafy Greens Board, and the California Strawberry Commission, which facilitated a number of projects discussed in the review, is gratefully acknowledged.

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UC Davis
UC Davis Previously Published Works
Title
Diversity, Pathogenicity, and Management of Verticillium Species
Permalink
https://escholarship.org/uc/item/4xr5g80x
Journal
Annual Review of Phytopathology, 47(1)
ISSN
0066-4286 1545-2107
Authors
Klosterman, Steven J
Atallah, Zahi K
Vallad, Gary E
et al.
Publication Date
2009-09-01
DOI
10.1146/annurev-phyto-080508-081748
Peer reviewed
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Diversity, Pathogenicity,
and Management of
Verticillium Species
Steven J. Klosterman,
1, ∗
Zahi K. Atallah,
2, ∗
Gary E. Vallad,
3
and Krishna V. Subbarao
2
1
USDA-ARS, Salinas, California 93905,
2
Department of Plant Pathology, University of
California, Davis, California 95616, and
3
University of Florida, Wimauma, Florida 33598;
email: kvsubbarao@ucdavis.edu
Annu. Rev. Phytopathol. 2009. 47:39–62
The Annual Review of Phytopathology is online at
phyto.annualreviews.org
This article’s doi:
10.1146/annurev-phyto-080508-081748
Copyright
c
 2009 by Annual Reviews.
All rights reserved
0066-4286/09/0908/0039$20.00
∗
Steven J. Klosterman and Zahi K. Atallah
contributed equally to this review and are to be
considered joint senior authors.
Key Words
taxonomy, species concept, host range expansion, host colonization,
seed transmission, host resistance
Abstract
The genus Verticillium encompasses phytopathogenic species that cause
vascular wilts of plants. In this review, we focus on Verticillium dahliae,
placing emphasis on the controversy surrounding the elevation of a
long-spored variant as a new species, recent advances in the analysis
of compatible and incompatible interactions, highlighted by the use of
strains expressing fluorescent proteins, and the genetic diversity among
Verticillium spp. A synthesis of the approaches to explore genetic diver-
sity, gene flow, and the potential for cryptic recombination is provided.
Control of Verticillium wilt has relied on a panoply of chemical and
nonchemical strategies, but is beset with environmental or site-specific
efficacy problems. Host resistance remains the most logical choice, but is
unavailable in most crops. The genetic basis of resistance to Verticillium
wilt is unknown in most crops, as are the subcellular signaling mech-
anisms associated with Ve -mediated, race-specific resistance. Increased
understanding in each of these areas promises to facilitate management
of Verticillium wilts across a broad range of crops.
39
Review in Advance first posted online
on April
22, 2009. (Minor changes may
still occur before final publication
online and in
print.)
Annu. Rev. Phytopathol. 2009.47. Downloaded from arjournals.annualreviews.org
by University of California - Davis on 06/22/09. For personal use only.

ANRV384-PY47-03 ARI 12 April 2009 14:58
INTRODUCTION
The genus Verticillium encompasses a cos-
mopolitan group of ascomycete fungi, includ-
ing several phytopathogenic species that cause
vascular wilts of plants. The two most notorious
species are V. dahliae and V. albo-atrum, which
cause billions of dollars in annual crop losses
worldwide (109). Yield losses in potato crops
may reach 50%, but are more commonly in
the range of 10–15% (114, 124, 125), whereas
in lettuce, losses can easily reach 100% (150).
The soil habitat of these species, the ability
of their survival structures to persist for years,
and their capacity to infect a bewildering
array of hosts make them chronic economic
problems in crop production. Four other
plant-pathogenic species historically associated
with the genus Verticillium are V. tricorpus,
V. nigrescens, V. nubilum, and V. theobromae
(13). The recent assignment of V. nigrescens
and V. theobromae to the genera Gibellulopsis
and Musicillium, respectively (170), reduced
the number of plant pathogenic species in the
genus Verticillium to four. In addition, both the
entomopathogenic V. lecanii and V. fungicola,
a pathogen of agaric basidiomycetes, were
assigned to the genus Lecanillium (169).
A variant of V. dahliae from horseradish,
first described by Stark (13) produces mi-
crosclerotia like V. dahliae but also conidia
significantly longer than the typical V. dahliae
strains, and thus was named V. dahliae var.
longisporum. This morphological difference and
other characteristics were considered sufficient
to elevate such strains into a new species,
V. longisporum (76). Erecting this new species
has been controversial and much effort over
the past decade has focused on resolving the
taxonomic, phylogenetic, and evolutionary
status of the long-spored crucifer strains. This
is the subject of the first part of this review. In
the context of this review, the taxonomic status
of Verticillium spp. is discussed relative to the
morphological and phylogenetic species con-
cepts, as described in numerous reviews (11, 86,
153, 154), and does not include the biological
species concept because it does not apply to this
genus. Verticillium spp. have no described sexual
stage.
Of the four remaining species in the genus,
V. dahliae is the suggested type species of the
genus (56) and also the more ubiquitous mem-
ber of the genus. It is the primary causal agent
of Verticillium wilt in temperate and subtrop-
ical climates (17, 69, 109) and is the focus of
this review. V. dahliae has great genetic plasticity
and is able to infect more than 200 plant species
(1), including high-value annual and perennial
crop plants, as well as landscape, fruit, and or-
namental trees and shrubs (17, 109). The list
of the hosts infected by V. dahliae is continually
expanding as disease outbreaks on new hosts
are identified (16, 43). One example of host
range expansion occurred in lettuce in coastal
California where entire crops have been lost
to Verticillium wilt (150, 161). The population
biology of V. dahliae remains the least under-
stood aspect of this ubiquitous phytopathogen.
Whereas strains of V. albo-atrum were divided
into two groups based on their virulence and
aggressiveness to lucerne (a.k.a. alfalfa, Med-
icago sativa), V. dahliae is divided based on veg-
etative compatibility into six groups (16, 71).
Nevertheless, vegetative compatibility groups
(VCGs) do not describe the overall genetic di-
versity among strains, gene flow, or the poten-
tial for recombination. They will, however, aid
in the deployment of resistant cultivars, pre-
venting pathogen introductions and exploring
the evolution of an agronomically important
group of phytopathogens.
The availability of cultivars tolerant to Ver-
ticillium wilt has reduced the disease to a minor
nuisance in some crops such as cotton. When
this type of resistance is compromised, Verti-
cillium wilt is likely to re-emerge as a signif-
icant production problem for such crops. Sev-
eral potato cultivars with improved resistance to
V. dahliae are available, in addition to wild and
cultivated accessions of Solanum tuberosum and
hybrids of Solanum spp. (32, 70). On tomato,
resistance to race 1 was overcome within a few
years after its introduction (109). Race 2 steadily
supplanted race 1 in various regions of the world
because of the extensive use of race 1-resistant
40 Klosterman et al.
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ANRV384-PY47-03 ARI 12 April 2009 14:58
cultivars (41). Two races were also described in
lettuce (65, 162), and germplasm with resistance
to race 1 was identified (65). However, there re-
mains no source of resistance to race 2 in either
tomato or lettuce. More importantly, resistance
in most crops is either scarce or unavailable,
making Verticillium wilt a significant chronic
problem in the production of these crops. Sub-
stantial economic losses caused by Verticillium
wilt are expected in the absence of fumigation
for high-value crops such as strawberry, potato,
cotton, tomato, and ornamentals (44, 125). For
the few crops in which resistance exists, the na-
ture of resistance has received little scrutiny. In
this review, we also cover studies exploring the
V. dahliae-lettuce interaction at the microscopic
level, providing a unique description of the in-
fection process from seedling to crop maturity.
We further define host resistance in a new light
based on studies using a fluorescently tagged
strain (163).
VERTICILLIUM TAXONOMY
Nees von Essenbeck erected the genus Verti-
cillium in 1816 (69, 109) based on its unique,
branched conidiophores, which form whorls
capped with flask-shaped and pointed phialides
carrying terminal conidia. Although a few
species of the genus Verticillium have been as-
sociated with ascomycetous teleomorphs (109,
170), V. dahliae, V. albo-atrum, and V. tricorpus
are solely anamorphic with no evidence of sex-
ual recombination or a meiosporic stage. In
1879, Reinke and Berthold first described wilt
on potato (Solanum tuberosum), and named the
causal agent V. albo-atrum (69, 109, 120). It
was not until 1913 that a second, morpholog-
ically distinct species causing wilt on dahlia
(Asteraceae family) was described by Klebahn,
and named V. dahliae (13, 69, 109, 120). Verticil-
lium spp. by convention, are identified based on
the type of resting structures produced, namely:
pigmented resting mycelium, pigmented mi-
crosclerotia, and chlamydospores. The two
most distinctive features separating V. dahliae
and V. albo-atrum are: (a) the production of
melanized microsclerotia as survival structures
by V. dahliae, in contrast to V. albo-atrum,
which produces melanized hyphae but no mi-
crosclerotia (13, 69, 109, 120), and (b) although
V. albo-atrum fails to grow in culture or wilt
plants at 30
◦
C, V. dahliae grows and infects un-
hindered at 30
◦
C (69, 109, 120). Even though
this information existed in the literature, the
taxonomic debate relative to the distinctive-
ness of these two species continued until the
late 1970s when V. dahliae was universally ac-
cepted as a species separate from V. albo-atrum
(53, 109). Subsequent phylogenetic studies have
clearly identified V. albo-atrum and V. dahliae as
distinct taxa (9, 13, 26, 117).
Based on host specificity, two clear subspe-
cific groups in V. albo-atrum are recognized.
Strains from alfalfa that cause severe symp-
toms on this host and also on numerous other
hosts, and strains from hosts other than alfalfa
that do not infect alfalfa or do so poorly (13,
31). This grouping is strongly supported both
by molecular markers (13) and VCG data (31,
61). A number of strains morphologically de-
scribed as V. albo-atrum cluster separately based
on the internal transcribed spacer region (ITS)
rDNA regions (99, 119), and thus were desig-
nated as V. albo-atrum Grp2. All other strains
of V. albo-atrum are referred to as Grp1 (13).
The resting structures and molecular mark-
ers such as random amplification of polymor-
phic DNA (RAPDs) and ITS sequences all dis-
tinguish these two groups (96, 119). Despite
recognizing that the differences are significant
enough to elevate Grp2 strains to a separate
species, they are currently only recognized as a
distinct operational taxonomic unit (96).
Unlike V. dahliae and V. albo-atrum, V. tricor-
pus is a successful soil saprophyte, which may
not be impeded in its germination and growth
by the absence of a potential host (69). Chlamy-
dospores, microsclerotia, and melanized hy-
phae serve as survival structures for V. tricorpus.
Because it is considered a weak pathogen on
many hosts, research has focused on the po-
tential for V. tricorpus to reduce the impact
of Verticillium wilt induced by V. dahliae or
V. albo-atrum. Co-inoculations of V. tricorpus
with V. dahliae in lettuce and artichoke, and
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Verticillium Species 41
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ANRV384-PY47-03 ARI 12 April 2009 14:58
V. dahliae or V. albo-atrum in potato have re-
sulted in a lower severity of Verticillium wilt or
potato early dying symptoms, compared with
plants inoculated with either of the latter two
species separately (116, 121).
CONTEMPORARY TAXONOMIC
CONTROVERSY
A variant strain of V. dahliae from horseradish
was described in 1961 by Stark (13). This vari-
ant produced microsclerotia but also conidia
that were significantly longer than the typ-
ical V. dahliae strains, and thus was named
V. dahliae var. longisporum. Similar strains caus-
ing Verticillium wilt have since been isolated
from cauliflower, oilseed rape, horseradish, and
other crops in North America and Europe (17,
18, 29, 49, 73, 76, 147, 172). These long-
spored strains produced conidia 7–9 μmin
length, nearly twice the size of typical V. dahliae
conidia (76). These long-spored strains actu-
ally produce a continuous distribution of size
classes (147) with the shorter conidia iden-
tical to V. dahliae in DNA content, whereas
the longer conidia contain approximately 1.75-
fold nuclear DNA content relative to most
short-spored V. dahliae (76). These long-spored
strains of V. dahliae were therefore described as
being “near-diploid” (76), and subsequently de-
scribed as amphihaploid (29). However, a strain
from Brussels sprouts in the United Kingdom
has been reported with short spores but higher
nuclear DNA content (76), as well as long-
spored strains with lower DNA content (147),
calling into question the relationship between
conidia length and DNA content.
Working exclusively with long-spored
strains from oilseed rape in Europe, Karapapa
et al. (76) considered the molecular and
other morphological differences with typical
V. dahliae strains distinctive enough to elevate
the long-spored, oilseed rape strains to a new
species, V. longisporum. It was further suggested
that the new species was host-specific and
borne out of an interspecific hybridization
between V. dahliae and a lucerne form of
V. albo-atrum (76). The elevation of this new
species has been questioned because the
studies by Karapapa et al. (76) were limited
to long-spored strains from European oilseed
rape and dismissed the type specimen used
by Stark (146) for the initial description of
V. dahliae var. longisporum as a recombinant
(13, 17, 26, 29). Erecting this new species
appears to be contrary to the morphological
and phylogenetic species concepts and has
generated focused research efforts to resolve
the taxonomic, phylogenetic, and evolutionary
status of strains assigned to this species.
A feature used to distinguish the crucifer
strain from the short-spored V. dahliae strain
is the in vitro morphological differences be-
tween their respective microsclerotia (73, 76).
Karapapa et al. (76) found the microsclerotia
produced in cultures of V. dahliae to be com-
pact, whereas those of the long-spored strains
were more diffuse and elongated and contained
dark hyphae. However, microsclerotia recov-
ered from infected crucifer crop tissues or from
soil have not revealed consistent morphological
differences among microsclerotia from long-
spored strains and those of typical V. dahliae
strains. The in vitro variability may be an ar-
tifact of the culture medium rather than a taxo-
nomic feature distinguising Verticillium species
(62).
There has been speculation that the am-
phihaploid crucifer strains of V. dahliae may
have arisen from hybridization between two or
more strains of V. dahliae or interspecific hy-
bridization between V. dahliae and V. albo-atrum
(28, 29). Interspecific hybridization has been
documented in certain fungi. For example, in-
terspecific hybridization of Botrytis aclada and
B. byssoidea gave rise to the allopolyploid B. alli
(145). Furthermore, strains of Verticillium spp.
carrying auxotrophic markers have been used to
obtain recombinant prototrophic strains in lab-
oratory studies, supporting parasexualism and
inter- and intraspecific hybridization in Verticil-
lium spp. (24, 63, 109). The high level of genetic
diversity observed in Verticillium spp. (10, 78)
could also be explained by the occasional gener-
ation of hybrid strains, which subsequently un-
dergo recombination and haploidization. The
42 Klosterman et al.
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Frequently asked questions

Q1. What are the contributions mentioned in the paper "Diversity, pathogenicity, and management of verticillium species" ?

In this review, the authors focus on Verticillium dahliae, placing emphasis on the controversy surrounding the elevation of a long-spored variant as a new species, recent advances in the analysis of compatible and incompatible interactions, highlighted by the use of strains expressing fluorescent proteins, and the genetic diversity among Verticillium spp. A synthesis of the approaches to explore genetic diversity, gene flow, and the potential for cryptic recombination is provided. 

Q2. What are the future works mentioned in the paper "Diversity, pathogenicity, and management of verticillium species" ?

An important area of future research also involves examining Verticillium spp. for the determinants of race specificity. Comparative genomic analyses will shed light on the potential origin of these strains. 3. Are there biological and/or cultural approaches, yet unidentified, that provide significant management of Verticillium wilt and may potentially reduce the need for chemical fumigants ? 

Q3. What is the mechanism by which organic amendments suppress pathogens?

The second mechanism by which organic amendments suppress pathogens is when soil pH is lowered upon coversion of NH4+ to nitrite (NO2−). 

Q4. What is the role of the hydrophobin gene in the development of the verticillium?

A hydrophobin gene, VDH1, is involved in microsclerotial developmentand spore viability in the plant pathogen Verticillium dahliae. 

Q5. Why are virgin lands virtually impossible to find?

Because Verticillium spp. are cosmopolitan, virgin lands to grow crops such as potato in North America are virtually impossible to find. 

Q6. What are the possible explanations for why broccoli is resistant to V. dahlia?

ontogenic changes in the type and levels of glucosinolates, structural components such as lignin, and phenolic compounds have been offered as possible explanations as to why broccoli is resistant to V. dahliae relative to cauliflower. 

Q7. What crops have been described as resistant to Verticillium wilt?

Genetic resistance to Verticillium wilt was described in alfalfa, cotton, potato, tomato, strawberry, sunflower, oilseed rape, lettuce (65, 109, 129), and other crops. 

Q8. Why is it possible that the long-spored strains are not the only hosts?

Because crucifers are not the only hosts for the long-spored strains (17, 149), it is possible that this isolation is driven by the infected hosts.