Potential of a Fusarium eumartii culture filtrate on the screening for

63
Euphytica 80: 63-69, 1994.
(~) 1994 Kluwer Academic Publishers. Printed in the Netherlands.
Potential of a Fusarium eumartii culture filtrate on the screening for wilting
resistance in potato
G r i s e l a L. B o t t a , M a r f a P. D i m a r c o , A l i c i a L. M e l e g a r i , M a r c e l o A. H u a r t e & C a r l o s A. B a r a s s i
Unidad lntegrada Balcarce (INTA-UNMP). CC 276, 7620 Balcarce, Argentina
Received7 February 1994; accepted 19 August 1994
Key words: Fusarium solani f. sp. eumartii, culture filtrate, electrolyte leakage, potato wilting, resistance, toxin,
Solanum tuberosum
Summary
Fusarium solani f.sp. eumartii Carp. Snyder and Hansen (Fusarium eumartii) is a soil inhabitant that induces the
so-called Potato Wilt and Stem End Rot disease. Prior to wilting, the pathogen induces peculiar small bronze spots
on the leaflets. Failure to isolate E eumartii from infected leaflets suggests the involvement of a toxin in the disease.
The fungus was grown in liquid Richard's medium and thereafter a filtrate was obtained dialyzing (MW cutoff
12,000-14,000) and sterilizing the culture by filtration (0.22 #m). Potato leaves treated with both the pathogen or the
filtrate showed symptoms of bronze spots and significantly higher electrolyte leakage when compared to controls.
Tomato leaves showed neither bronze spots nor electrolyte leakage after plant inoculation with the pathogen or with
the filtrate treatment. Both, the absence of visible symptoms and the lack of electrolyte leakage in tomato could be
associated to a certain degree of host specificity of the F. eumartii filtrate towards potato. The filtrate also induced
symptoms similar to infections by E eumartii in adult plants and in vitro plantlets of cultivars Huinkul MAG and
Kennebec. Callus responses to the filtrate were related to responses of the cultivars to the pathogen in greenhouse.
These results show the potential of the culture filtrate of E eumartii for use in screening for wilting resistance.
Introduction
Potato Wilt and Tuber Stem End Rot caused by Fusarium solani f.sp. eumartii Carp. Snyder and Hansen
(Fusarium eumartii) is endemic in Argentina and was
reported to occur in some regions of Chile, Brazil and
USA (Calderoni, 1978; Gerlach & Nirenberg, 1982).
Yield losses close to 40% were registered in the susceptible cultivar Huinkul MAG under a severe epidemic
outbreak in Argentina (Malamud, 1970).
The pathogen is a soil inhabitant that infects potato
plants through roots and seed-tuber pieces and its invasion is restricted to the stem base, stolons and tuber
stem end. Prior to wilting, the pathogen induces small
bronze spots on the leaflets. In tubers, the fungus produces stem end dry rot and brown discoloration of the
vascular ring.
Failure to isolate E eumartii from infected leaflets
and vascular ring suggests the involvement of a toxin
in the disease (Goss, 1924; Thomas, 1949). However,
neither the isolation of such a toxin nor the use of culture filtrates for breeding purposes have been reported
so far.
The objectives of this study were a) to obtain a F.
eumartii filtrate able to reproduce in vitro the symptoms
observed in vivo after fungal infection, b) to determine
the effect of the filtrate on membrane permeability of
leaves of the susceptible cultivar Huinkul MAG and its
specificity towards potato and, c) to determine whether
cultivar responses to the filtrate are related to cultivar
responses to the pathogen.
Materials and methods
Fungal culture and filtrate preparation
A virulent strain of E eumartii (strain A) was cultured
on 2% potato dextrose agar (PDA), incubated 1 wk at
250 C in the dark and 2 wk at 180 C 4- 20 C under
64
continuous light (60/~E m -2 s-l). A 0.5 cm diameter agar disc with fungal growth was transferred to a
250 ml flask with 100 ml of liquid Richard's medium containing 10.0 g KNO3, 5.0 g KH2PO4, 2.5 g
MgSO4.7H20, 0.02 g FeC13 and 50.0 g sucrose per
liter of distilled water (The Commonwealth Mycological Institute, 1985). Cultures were incubated 4 wk
at 220 C + 20 C in the dark with shaker agitation at
180 r min- 1 during 9 h per day. Cultures were filtered
through Whatman N O 1 paper and dialyzed (MW cutoff
12,000-14,000) against 11 of distilled, deionized water
during 24 h at 4 ° C. Initial and final conductivities were
17.4 and 4.5 mmhos cm-1 at 250 C respectively, both
in culture and in control filtrates. After pH adjustment
to 7.0 with 1 M KOH, the liquid was sterilized by filtration with a SARTORIUS SM 16510 filter unit, provided with a GCFMS 9043 MM pre-filter and a 0.22 #m
filter. Non-inoculated Richard's medium was equally
dialyzed and filtered to be used in control treatments.
Filtrates were stored at 40 C in the dark until used.
When required, conidial suspensions were obtained as
follows: mycelia were scraped from PDA growth and
suspended in sterile, distilled water. Conidia washings
were accomplished by successive centrifugations and
re-suspensions in sterile distilled water. Final concentration was 3.0 x 10 6 conidiam1-1.
Bioassay
Non-inoculated whole leaves of Solanum tuberosum
cultivar Huinkul MAG and Lycopersicum esculentum
cultivar Plautaco INTA, were used to study the effect
of the filtrate on membrane permeability. Potato leaves
were detached from the middle portion of 8-wk-old
healthy plants growing in greenhouse at 180 C + 80 C.
Only one leaf was taken from each plant. Tomato leaves
were excised from seedlings with two leaves growing
in greenhouse under the conditions mentioned above.
Only leaf petioles were immersed either in F. eumartii
filtrate or in Richard's filtrate and incubated at 22 ° C -42 ° C, 16 h light (90 #E m - 2 s- 1)/8 h dark and humidity
close to 100%. After 48 h of incubation, leaves were
examined for presence of bronze spots and modified
cell permeability. Changes in cell permeability were
determined with a conductivity method which measures ion leakage (Sukumaran & Weiser, 1972). All
the leaflets of each leaf were placed in an individual flask containing 30 ml of distilled, deionized water
and incubated at 22 ° C with shaker agitation at 180 r
min -1. After 1 h shaking, initial liquid conductance
was measured with a Metrohm 644 Conductometer.
Leaflets were frozen at - 600 C during 24 h to kill the
tissues, re-immersed in the original liquid and shaken for 1 h. Final conductivity of the liquid was measured. Percent conductivity, calculated as the ratio of
the initial to the final conductivities, was expressed as
percent of electrolyte leakage. Electrolyte leakage was
also measured in leaves with bronze spot symptoms of
cultivar Huinkul MAG inoculated with F. eumartii and
in symptomless leaves of controls. In all the experiments each leaf represented one sample. No more than
one sample was taken from each plant. Means were
compared by Duncan's test (p < 0.025). Confidence
limits were constructed for the comparisons.
Potato callus and in vitro plants
Potato stem cuttings from in vitro plants were cultured in the minimal organic medium of MurashigeSkoog (Murashige & Skoog, 1962), containing 30.0 g
sucrose, 8.0 g agar, 2.5 g gibberellic acid and 0.002 g
calcium pantothenate per liter of distilled water, and
incubated at 22 ° C, 18 h light (70/_rE m - 2 s- 1)/6 h dark.
Multiplication was carried out every six weeks.
Leaflets of potato cultivars Huinkul MAG, Kennebec and Russet Burbank were used for callus production. Leaf discs (10 mm diam) were successively
immersed in 70% ethanol for 10 sec and in NaOC1
(2% C1) for 15 min, rinsed three times in sterile distilled water and dried on sterilized filter paper. Discs
were cultured on Murashige-Skoog minimal organic
medium, plus 30.0 g sucrose and 8.0 g agar per liter
of distilled water. Medium was supplemented with
2 mg l-1 naftalen acetic acid (NAA) and 0.5 mg l6-benzilaminopurine (BPA) (Ochatt & Caso, 1986) for
cultivars Huinkul MAG and Russet Burbank, or 5 mg
1-1 NAA and 1 mg 1-1 BAP for cultivar Kennebec
(Botta, 1992). Cultures were incubated at 220 C, 18 h
light (70 #E m -2 s-1)/6 h dark.
Greenhouse experiments
Three sets of experiments are included in this section:
one was designed to check strain virulence and specificity towards potato and the other two, to evaluate
disease incidence (DI) after inoculation with conidia
and after inoculation with the fungal filtrate, respectively.
Plants of S. tuberosum cultivar Huinkul MAG and
L. esculentum cultivar Plautaco INTA growing in autoclaved soil, were inoculated with E eumartii. Inoculation was carried out at stages of four and two devel-
65
80 C. Leaves were periodically examined for the presence of bronze spots.
For the following experiments, three-week-old in
vitro plants of cultivars Huinkul M A G and Kennebec
were individually transplanted into pots containing
soil sterilized with methyl bromide (450 g/1.87 m 3
of soil). Plants grew in the greenhouse for 30 days
at 180 C 4- 80 C. Prior to inoculation, plants were
removed and their roots washed with distilled water.
In one experiment, roots were immersed in a conidial suspension containing 3.0 × 10 6 conidia m1-1.
Treatments were arranged in a completely randomized design with four replications including 20 plants
each. In the other experiment roots were immersed in
the filtrate. Treatments were arranged in a completely randomized design with five replications including
20 plants each. Plants with root immersion in distilled
water or in Richard's filtrate were used as controls in
the first and in the second experiment, respectively.
After treatment, plants were transplanted to the same
pots. Disease incidence was recorded as the number
of wilted plants after 50 days from inoculation. Percentages transformed by the arcsin square root were
subject to analysis of variance. Means were compared
by Waller's test (p _< 0.05).
In vitro e x p e r i m e n t s
Fig. 1. Early symptoms on potato leaves infected by E eumartii or
treated with F. eumartii filtrate. Interveinalbronze spots scattered on
leaflets from (A) F. eumartii - infected potato plants, and (B) leaves
treated with E eumartii filtrate.
oped leaves in potato and tomato, respectively. Prior to inoculation, plants were removed, their roots
washed with distilled water and finally immersed for
5 min in distilled water containing 3.0 × 10 6 conidia
m l - 1. Plants with root immersion in autoclaved inoculum were included as controls. Inoculated plants were
transplanted to the original pots and grown at 180 C 4-
Four-week-old in vitro plants of cultivars Huinkul
MAG and Kennebec individually cultured in test tubes,
were inoculated with 0.5 ml of filtrate injected in the
medium near the roots. Plants inoculated with the same
amount of Richard's filtrate were included as controls.
Plants were incubated at 220 C, 18 h light (70 # E m -2
s - 1)/6 h dark. Treatments were arranged in a completely randomized design with five replications, including
fifteen plants each. Disease incidence was recorded as
the number of wilted plants after 12 days from inoculation. Percentages transformed by the arcsin square
root were subject to analysis of variance. Means were
compared by Waller's test (p < 0.05).
Ten-day-old callus of cultivars Huinkul MAG, Kennebec and Russet Burbank selected for uniform size,
were cultured in petri dishes containing the medium
and the growth regulators used for callus production.
In one experiment, calli were inoculated by applying
0.05 ml of the filtrate on the surface. Treatments were
arranged in a completely randomized design with five
replications, each including five petri dishes and four
calli per dish. After 12 days, calli were scored on a
scale of 0-3, where 0 = absence of symptoms; 1 =
66
loss o f turgor and necrosis restricted to the inoculation
point; 2 = loss o f turgor and partial necrosis; 3 = generalized necrosis. Disease severity (DS) was expressed
as: DS = ~(i=0-i=3)Xi(ni)/Xt(n3), where ni = score in
the severity scale; Xi = number of callus in class ni; Xt
= total number o f callus. In other experiment, calli were
cultured on media containing the filtrate. Filtrate was
added to the previously autoclaved and cooled medium
to reach final concentrations o f 10, 25 and 50% (v:v).
Treatments were arranged in a completely randomized
design with three replications, each including 62, 96
and 110 calli o f cultivars Russet Burbank, Huinkul and
Kennebec, respectively. No more than five calli per
dish were included. After 30 days, calli were scored
on a scale o f 0 - 2 , where 0 = normal growth and green
colour; 1 = impaired growth and restricted necrosis; 2
= absence o f growth and generalized necrosis. Disease
severity was expressed as: DS = )](i=0_i=2)Xi(ni)/X
t(n2). In both experiments, calli treated with equivalent
amounts and concentrations o f Richard's filtrate were
used as controls. Cultures were incubated at 220 C,
18 h light (70 # E m - 2 s - l ) / 6 h dark. Means of disease severity were compared with Waller's test (p <
0.05).
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Results
Attempts to experimentally infect tomato plants with
the conidial suspension o f E eumartii under greenhouse conditions, were unsuccessful. Neither bronze
spots nor wilting were observed after 30 days from
inoculation. Potato leaves from cultivar Huinkul M A G
showed bronze spots as early as 10 days from inoculation (Fig. 1A), and the harvested potatoes from infected
plants developed the characteristic brown discoloration
o f the vascular ring (data not shown).
Fig. 2. Electrolyte leakage in leaves of (a) Solanum tuberosum
cultivar Huinkul MAG and (b) Lycopersicum esculentum cultivar
Plautaco INTA. Measurements were done in: leaves with bronze
spots, detached from plants inoculated with conidia of Fusarium
eumartii I---1 (n = 9); healthy leaves detached from uninoculated
plants ~
(n = 13); healthy leaves detached from uninoculated
plants, and treated with the filtrate ofE eumartii during 48 h ~ (n
= 9), • (n = 12); healthy leaves detached from uninoculated plants,
and treated with Richard's filtrate during48 h ~ (n = 6), ~.'.~ (n =
12). Means were compared with Duncan test (p = 0.025). Electrolyte
leakage values in potato leaves either inoculated or treated with the
filtrate are significantlyhigher than those of the respective controls.
Bioassay
The filtrate o f F. eumartii induced typical bronze spots
on potato leaves after 48 h treatment (Fig. 1B). No visible symptoms were noticeable in tomato leaves after
48 h treatment with the filtrate (data not shown).
Electrolyte leakage percentages in potato leaves
inoculated with F. eumartii or treated with the filtrate,
were significantly higher (p < 0.025) than those o f the
corresponding controls (Fig. 2a). Electrolyte leakage
in leaves o f tomato treated with the filtrate was not significantly different (p < 0.025) from that o f the control
(Fig. 2b). No changes in membrane permeability were
detected in leaves o f tomato plants inoculated with F.
eumartii when compared to control leaves (data not
shown).
Inoculation o f plants with conidia or filtrate in
greenhouse and in vitro
Both, conidia and filtrate o f F. eumartiiinduced wilting
in cultivars Kennebec and Huinkul M A G in greenhouse
and in vitro conditions. In all the experiments, controls
remained healthy. Cultivar Kennebec had a significantly (p < 0.05) lower average DI than cultivar Huinkul
67
0
60
100
O"
~ ' O'
80
~20
,
10
10"
20
0 Conidla
Filtrate
0
Treatment
Fig. 3. Effect of conidiaand filtrateof Fusarium eumartii on plants
(a) and callus (b) of cultivars Huinkut MAG I----1and Kennebec
under greenhouseand in vitro. In all the experimentscultivar
Kennebecregistered significantly(p = 0.05) lowerwiltingincidence
or disease severityin callusthan cultivarHuinkulMAG.Wiltingincidence percentagesare means of 75-100 plants per cultivar.Disease
severity percentagesare means of 100 callus per cultivar. In all the
experimentsmeans were comparedwith Wallertest (p = 0.05).
M A G regardless of the inoculation method used (Fig.
3a). In the greenhouse, cultivar Huinkul MAG registered 46% of wilted plants after the treatment with
either conidia or filtrate. In cultivar Kennebec, the incidences were 31% and 23% when plants were treated
with conidia and filtrate, respectively. In vitro plants
treated with the filtrate, registered 49% and 28% of
incidence in cultivars Huinkul and Kennebec, respectively. Necrotic lesions in roots were evident in the
in vitro plants treated with the filtrate as early as 7
days after treatment. These results agree with previous
observations in the field under natural and artificial
inoculations with E eumartii (Calderoni, 1954; Escande & Radtke, 1973).
10
2$
$0
Filtrate concentration (%)
Fig. 4. Diseaseseverityin callus of cultivarsHuinkul E"-I, Russet
Burbank ~ and Kennebec• cultured on media containingdifferent concentrationsof Fusarium eumartii filtrate. Disease severity
percentages are means of 62, 96 and 110 callus of cultivarsRusset
Burbank, Huinkul and Kennebec,respectively. Cultivar Kennebec
registered significantlylower (p = 0.05) disease severity than cultivars Huinkul MAG and Russet Burbank. Means were compared
with Wallertest (p = 0.05).
Inoculation o f callus with the filtrate
Average DSs registered in calli inoculated with the E
eurnartii filtrate were significantly (p < 0.05) lower
in cultivar Kennebec than in cultivar Huinkul M A G
regardless of the inoculation method used (Figs 3b and
4). In both experiments controls had no symptoms. In
surface inoculation, calli had DSs of 57% and 25%
for cultivars Huinkul M A G and Kennebec, respectively (Fig. 3b). When culture of callus on media with the
filtrate was used, the interaction between cultivar and
filtrate concentrations was not significant (p _< 0.05).
Every increase in the filtrate concentration resulted in
68
a significant increase of the average DSs of cultivars
Kennebec, Huinkul MAG and Russet Burbank (Fig.
4). In both experiments, cultivar Russet Burbank registered DSs that did not differ from those of cultivar
Huinkul MAG.
Discussion
Fusarium eumartii has been reported to infect other S. tuberosum-related species (Hooker, 1980), and
several leguminous plants (Trapero-Casas & JimrnezDfaz, 1985) under experimental conditions. However, studies done in Argentina show S. tuberosum as
the specific host of F. eumartii (Carrera, 1972). Initial symptoms caused by F. eumartii are different from
those of other wilt diseases and are easily detected.
The onset of small light green areas between the veins
of the leaflets giving a mottled appearance is followed
by a yellowing of these areas. This is often accompanied by small irregular bronze spots on the upper
surface of the leaf (Goss, 1924). Some Argentinian potato cultivars develop the typical bronze spots
without prior formation of pale areas, a characteristic
which is useful in the diagnosis of F. eumartii (Escande et al., 1984). Our attempts to infect tomato plants
with a virulent strain of F. eumartii were unsuccessful. However, the susceptible S. tuberosum cultivar
Huinkul MAG showed the characteristic bronze spots
followed by wilting (Fig. 1A) and brown discoloration
of the vascular ring in tubers (data not shown). On
the other hand, electrolyte leakage has been related
to changes in permeability (Wheeler, 1976) and has
been regarded as one of the earliest host responses to
a variety of plant pathogens (Misaghi, 1982). However, only the host-selective Helminthosporium victoriae
toxin (HV) causes permeability changes in oat tissues
identical to those caused by the pathogen in experimentally infected plants (Wheeler & Black, 1963).
In our study, both the E eumartii conidia and filtrate
increased ion leakage in potato leaves while no significant changes appeared in tomato leaves (Fig. 2).
Both the absence of visible symptoms and the lack of
electrolyte leakage in tomato might be associated to
a certain specificity of the E eumartii filtrate towards
potato. Or at least, these data suggest the absence of
non-specific substances in the filtrate that have been
related to changes in membrane permeability in infected tomato plants (Iacobellis & Bottalico, 1981).
None of several evaluating points proposed to determine whether a toxin has a role in plant disease, has
any importance by itself. Yet, in combination confidence may increase (Yoder, 1980). Our experiments
have shown that cultivar responses to the culture filtrate of E eumartii were related to responses of the
cultivars to the pathogen. We found similar changes
in ion leakage both in infected potato plants and in
filtrate-treated leaves having the same visible symptoms (Figs 1 and 2). Moreover, we found no visible
symptoms or increased ion leakage both in inoculated
tomato plants and filtrate-treated tomato leaves (Fig.
2b).
The use of toxic metabolites instead of the pathogen
to evaluate plant response might be valuable, mainly
when the disease expression is highly influenced by
the environment. In the case of E eumartii wilting,
symptom onset is favoured by dry soil. Although many
investigators succeeded in the selection in culture for
resistance to a pathogen toxin, others found that host
response to the toxin was not correlated to pathogenicity (Daub, 1986). In our study, adult plants and in vitro
plantlets exposed to the filtrate showed symptoms similar to infections by E eumartii (Fig. 3a). Moreover,
plant and callus responses to the filtrate were related
to responses of the cultivars to the pathogen in greenhouse (Figs 3 and 4). These results show the potentiality of the culture filtrate for use in the screening for
resistance to F. eumartii. A system in which both the
filtrate and in vitro plants may be used would enable
testing large numbers of plants under conditions that
guarantee symptom expression.
Recovery of disease resistant plants by in vitro
selection at cellular level using partially purified toxin
or culture filtrate produced by a plant pathogen, has
been reported (M. Daub, 1986; Arcieni et al., 1987;
Frame et al., 1991). Further purification of the filtrate
in order to identify toxic metabolites of E eumartii
involved in pathogenicity, should be performed. The
potential use of the filtrate of E eumartii in tissue culture to determine structural and biochemical barriers
involved in defense mechanisms and its usefulness for
resistance induction in cells and tissue cultures, has to
be investigated.
Acknowledgements
This work was partially supported by a grant from Consejo Nacional de Investigaciones Cientfficas y Trcnicas
(CONICET), given to C.A. Barassi. The authors thank
Mr. Oscar Gerpe for helping in the experimental work,
69
and Dr. Alberto Escande for critical reading of this
paper.
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