Aim of study
: To assess the impact on two mycoviruses recently described in F. circinatum mitovirus 1 and 2-2 (FcMV1 and FcMV2-2) on i) mycelial growth, ii) spore germination and iii) relative necrosis.
Material and methods
: Fourteen monosporic strains of
F. circinatum
(one of each pair infected with mycoviruses and the other without them) of the pathogen with and without viruses were selected for the assay. The statistical analysis, were a linear mixed model of analysis of variance considering one between-subjects factor (isolate) and one within-subjects factor with four levels (1=without viruses, 2=only virus FcMV1, 3=only virus FcMV2-2 and 4=both viruses).
Main results
: Colony growth rates of
F. circinatum
isolates were significantly reduced in presence of mycoviruses (
p
=0.002). The spore germination was also reduced in the
F. circinatum
isolates containing mycovirus as compared to mycovirus-free isolates (
p
<0.001). No significant differences in lesion lengths caused by
F. circinatum
were found in relation to the presence/absence of mycovirus (
p
<0.61).
Research highlights
: Reduction of the percentage of spore germination in the isolates of
F. circinatum
with mycovirus infections, as compared to free isolates, provides indications of reduction of metabolic activity and plant physiology are discussed. The lack of significant differences found in the length of the lesions caused by
F. circinatum
with respect to the presence/absence of mycovirus, indicates that further studies with a larger number of variables are required
Authors´ contributions:
Conceived, designed the experiment and performed the experiments: JAFP, EJMA, PMA and JJD. Analyzed the data: VP. Wrote the paper: JMG, JAFP and JJD. All authors reviewed the paper.
The Ministry of Economy and Competitiveness of the Government of Spain (Projects AGL2012-39912 and AGL2015-69370-R); COST Action FP1406 PINESTRENGTH; Academic Mobility program for Inclusive Development in Latin America (AMIDILA) / Erasmus Mundus Action 2 in partnership with Bluefields Indian & Caribbean University (BICU) – Nicaragua (Scholarship to JAFP); Portuguese Foundation for Science and Technology (FCT) supported JMG (Post doc grant - SFRH/BPD/122928/2016).
Competing interests:
The authors have declared that no competing interests exist.
Introduction
Fusarium circinatum
(teleomorph
Gibberella circi- nata
Nirenberg & O’Donnell) is an ascomycetous fungus that causes pine pitch canker (PPC) disease in
Pinus
spp. and other conifers such as
Pseudotsuga menziesii
(
Barnard & Blakeslee, 1987
;
Dwinell
et al.
, 1998
). The pathogen was first detected in 1945 in
Pinus virginiana
Mill in the southeastern United States (
Hepting & Roth, 1946
). Since then, it has continued to expand throughout the world and has been found infecting different conifer species in Mexico (
Guerra-Santos, 1998
), Haiti (
Hepting & Roth, 1953
), Japan (
Kobayashi & Muramoto, 1989
), South Africa (
Viljoen
et al.
, 1994
,
Coutinho
et al.
, 2007
), Chile (
Wingfield
et al.
, 2002
), Korea (
Cho & Shin, 2004
), Uruguay (
Alonso & Bettucci, 2009
), Colombia (
Steenkamp
et al.
, 2012
) and, more recently, in Brazil (
Pfenning
et al.
, 2014
). In Europe, the pathogen has been also recorded in Spain (
Dwinell, 1999
;
Landeras
et al.
, 2005
), France (
EPPO, 2006
), Portugal (
Bragança
et al.
, 2009
) and Italy (
Carlucci
et al.
, 2007
).
PPC fungus causes severe symptoms in adult trees (
e.g.
wilting and reduced growth) with frequent formation of cankers producing abundant amounts of resin that usually appears on the trunk or thicker branches. The lesions damage pine trees and make them susceptible to windthrow and can even kill the trees by interrupting the sap flow (
Hepting & Roth, 1946
). The fungus also causes damping off in seedlings, leading to mortality rates of up to 100% (
Martinez-Álvarez
et al.
, 2014a
). This necrotrophic fungus is thus considered the most important pathogen of pine seedlings on a global scale (
Wingfield
et al.
, 2008
).
The presence of the pathogen in Spain has caused high economic losses because of the ban on planting susceptible species, as well as the high costs of monitoring the disease and implementing treatments aimed at eradication (
BOE, 2006
,
2010
). However, no effective treatments have been development in the field and efforts have mainly focused on nursery treatments (
Gordon
et al.
, 2015
). Biological control is emerging as one of the most promising options for limiting the effects of the disease (
Sánchez-Fernández
et al.
, 2013
).
The use of mycoviruses is considered one of the most promising strategies for biological control of forest diseases (
Muñoz-Adalia
et al.
, 2016a
). In fact, some mycoviruses can be used as biocontrol agents by promoting hypovirulence in their host, with the best-known case in forest pathology being the chestnut blight caused by
Cryphonectria parasitica
(
Carey
et al.
, 2005
;
Zamora
et al.
, 2012
). Mycovirus infections can lead to induction of a cryptic state (asymptomatic), reduction of mycelial growth, change in the colour of the colony or even alteration of the specific structure formation (
Rodríguez-García
et al.
, 2014
). The effects of mycoviruses on spore production and pathogenesis have also been widely reported (
Kazmierczak
et al.
, 1996
;
Wang
et al.
, 2014
, Zamora
et al.
, 2016), indicating the potentially damaging effects of the viruses on plant health. Three different species of the genus
Mitovirus
that infect
F. circinatum
have recently been identified as putative members of Narnaviridae (genus Mitovirus) and designated
Fusarium circinatum
mitovirus 1, 2-1 and 2-2 (FcMV1, FcMV2-1 and FcMV2-2) (
Martínez-Álvarez
et al.
, 2014a
;
Vainio
et al
., 2015
;
Muñoz-Adalia
et al.
, 2016a
).
The main aim of the study was to evaluate how mycoviruses affect the pathogenicity of
F. circinatum.
For this purpose, we assayed colony growth, spore germination and pathogenicity of isolates on
Pinus radiata
D. Don seedlings.
Material and methodsFungal isolates
Seven
F. circinatum
isolates were selected for the study. Two paired monosporic cultures (mycovirus-infected and mycovirus-free) were used per isolate (Table 1). The presence of different mycovirus strains in each isolate was reported in a previous study by our research group (
Vainio
et al.
, 2015
). In the present study, the presence or absence of mycoviruses (FcMV1 and/or FcMV2-2) was confirmed according to
Martínez-Álvarez
et al.
(2014b)
. For in vitro experiments, an isolate (FcCa06) previously confirmed as mycovirus-free and pathogenic (
Martínez-Álvarez
et al.
, 2012
,
2014a
) was also used.
Results of tests of Fusarium circinatum isolates (seven isolates, two monosporic cultures/isolate): origin;
mycovirus infection (- =absence of mycovirus and + =presence of mycovirus); host (Pp=Pinus pinaster, Pr=Pinus
radiata); MAT: mating-type; spore germination (SG), expressed as a percentage; area of the fungal colony (AFC) in mm2;
relative necrosis length (LRN) in mm.
In vitro experiments: mycelial growth and spore germination
where D= diameter. A sample of 100 µL of conidial suspension of
F. circinatum
isolates was grown in Potato Dextrose Broth (PDB) supplemented with 5 g/L streptomycin sulphate (PDBS) (
Martinez-Alvarez
et al.
, 2012
). Three flasks were prepared for each isolate. The spores were incubated for 6 hours under stirring at 180 cycles/min at 25° C in darkness and at 60% humidity. Three aliquots (each 10 µL) of the spore suspension were removed from each flask for evaluation of germination (200 conidia in each sample,
i.e.
a total of 1,800 conidia were scored). The spore suspensions were examined under a light microscope (Nikon E600) with a 40x lens. A hemocytometer was used to count the number of germinated spores as the grid helped to avoid counting the same spore more than once.
In vivo experiments: pathogenicity tests
Pathogenicity tests were performed with two-year-old seedlings of
P. radiata
from Galicia, Spain. The plants were raised individually in root trainers (cells of 200 mL) and incubated in a growth chamber at 21º C with a 16-h photoperiod, following a completely randomized design.
The total length of the seedlings, the root collar diameter and the length of necrotic damage were measured in all seedlings. In order to obtain a more accurate view of the extent of the necrosis, the stems were split lengthways with a scalpel. To avoid the effect of the size of the seedlings, the relative necrosis was estimated as the ratio between length of the necrotic lesion and total length of the seedling.
Statistical analysis
Mycelial growth. For the statistical analysis, we used a linear mixed model of analysis of variance with repeated measurements, considering four between-subject factors and one within-subject factor with seven levels. The mathematical formulation of the model was as follows:
where
j
=0 (experimental unit with an isolate) or 1 (control without an isolate);
k
=0 (isolate without virus 1) if
j
=1 or
k
=1 (isolate with mycovirus1) if
j
=0;
l
=0 (isolate without virus 2) if
j
=1 or
l
=1 (isolate with mycovirus 2) if
j
=0;
i
=1,..,15 for the isolates with
j=k=l=
0 if
i
=1,..,5,
j
=
k
=0 and
l
=1 if
i
=6,..,9,
k
=1 and
j
=
l
=0 if
i
=10,..,13,
j
=0 and
k
=
l
=1 if
i
=14,
j
=1 and
k
=
l
=0 if
i
=15;
n
=1,..,7 for the replicates of the experimental units and
t
=2,..,7 for the repeated measurements (from day 2 to day 7).
Spore germination. For the statistical analysis, we used a linear mixed model of analysis of variance with repeated measurements, considering four between-subject factors and one within-subjects factor with three levels. The mathematical formulation of the model was given as follows:
where
j
=0 (experimental unit with an isolate) or 1 (control without an isolate);
k
=0 (isolate without mycovirus 1) if
j
=1 or
k
=1 (isolate with mycovirus 1) if
j
=0;
l
=0 (isolate without mycovirus 2) if
j
=1 or
l
=1 (isolate with virus 2) if
j
=0 ;
i
=1,..,15 for isolates with
j=k=l
=0 if
i
=1,..,5,
j
=
k
=0 and
l
=1 if
i
=6,..,9,
k
=1 and
j=l
=0 if
i
=10,..,13,
j
=0 and
k=l
=1 if
i
=14,
j
=1 and
k=l
=0 if
i
=15;
n
=1,..,3 for the replicates of the isolates and
t
=1,2,3 for the repeated measurements (1 for 6 hours, 2 for 12 hours and 3 for 24 hours).
Relative necrosis. For the statistical analysis, we used a linear mixed model of analysis of variance with three between-subject factors. The mathematical formulation of the model was given as follows:
where
k
=0 (isolate without mycovirus 1) or 1 (isolate with mycoirus1);
l
=0 (isolate without mycovirus 2) or 1 (isolate with mycovirus 2);
i
=1,..,14 for the isolates with
k=l
=0 if
i
=1,..,5,
k
=0 and
l
=1 if
i
=6,..,9,
k
=1 and
l
=0 if
i
=10,..,13, and
k=l
=1 if
i
=14;
n
=1,..,27 for the replicates. Finally, we used an individual pairwise t-test for all comparisons between the least square means. All tests were carried out with SAS® 9.4 statistical software (Foundation SAS 9.1.3, 2008).
Results and discussionEffect of Fusarium mycoviruses on colony growth
In vitro growth rates of the
F. circinatum
isolates varied significantly depending on the presence/absence of mycoviruses (F=6.03,
p
=0.002). In particular,
F. circinatum
isolates infected with micoviruses, both single infections with FcMV1 and FcMV2-2 (t=2.39,
p
=0.02 and t=3.63,
p
<0.001, respectively) and co-infection with FcMV1 + FcMV2-2 (t=4.27,
p
<0.001), yielded lower growth rates than mycovirus-free
F. circinatum
isolates (Fig. 1A). There were no significant differences between mycovirus-infected
F. circinatum
isolates in relation to the type of mycovirus (FcMV1, FcMV2-2 or co-infection) (Fig. 1A). A reduction in the mycelial growth of different pathogens infested with mycoviruses has been reported to be related to hypovirulence in fungi in several studies (
Bottacin
et al.
, 1994
;
Ahn & Lee, 2001
;
Castro
et al.
, 2003
;
Robin
et al.
, 2010
;
Rodríguez-García
et al.
, 2014
;
Zheng
et al.
, 2014
;
Yu
et al.
, 2015
). Nevertheless, the effect on mycelial growth may also be determined by the species (
Vainio
et al.
, 2012
;
Hyder
et al.
, 2013
), isolate (
Hunst
et al.
, 1986
;
Rodríguez-García
et al.
, 2014
;
Yu
et al.
, 2015
) temperature (
Vainio
et al.
, 2010
;
Bryner & Rigling, 2011
;
Romeralo
et al.
, 2012
; Zamora
et al.
, 2016) or the type of mycovirus (
Cryphonectria parasitica
CHV1, mycovirus hypovirulences). These factors, among others, may explain the inconsistencies in the findings of different studies. The conditions of the experiment appear to be related to the effect of the virus, and use of the same stains as those used by
Muñoz-Adalia
et al.
(2016b)
thus failed to yield any effect of mycoviruses on the mycelial growth of
F. circinatum
.
Effect of mycovirus infection on mycelial
growth, mm2 (A) and on germination, % (B) in Fusarium
circinatum. Data were analyzed by the restricted maximum
likelihood (REML) for fixed effects. Lowercase letters (ab)
denote significant differences (T-Student test, p<0.05).
Values shown are means and standard errors.
Effect of
Fusarium
mycoviruses on spore germination
A large reduction in spore germination was observed in the mycovirus-infected isolates of
F. circinatum,
relative to mycovirus-free isolates (F=28.21,
p
<0.001). Again, mycovirus-infected isolates, both single infections with FcMV1 and FcMV2-2 (t=28.76,
p
<0.001 and t=13.18,
p
<0.001, respectively) and co-infection with FcMV1 + FcMV2-2 (t=8.81,
p
<0.001), yielded lower germination rates than mycovirus-free isolates (Fig. 1B). No significant differences were observed in relation to the mycovirus. Some studies have demonstrated that the presence of hypovirulence-causing mycoviruses also reduces sporulation (
Kazmierczak
et al.
, 1996
,
Moleleki
et al.
, 2003
;
Robin
et al.
, 2010
; Zamora
et al.
, 2016). Thus,
Ihrmark
et al.
(2004)
observed that the frequency of germination of basidiospores was reduced by the presence of mycoviruses. However, to our knowledge no specific research has been carried out to study the effect of mycoviruses on spore germination in ascomycetes.
Effect of
Fusarium
mycoviruses on pathogenicity
The pattern observed in the
in vivo
experiment was not consistent with the findings of the
in vitro
assays (both mycelial growth and spore germination). In fact, no significant differences in the length of lesions caused by
F. circinatum
were found in relation to the presence/absence of mycovirus (F=0.62,
p
<0.61) (Table 2). Although necrosis has traditionally been considered an indicator of the disease progress, because
F. circinatum
kills stem tissue during colonization (
Glazebrook
et al.
, 2005
), recent research has demonstrated that
F. circinatum
can colonize the plants without causing necrosis, at least at the early stages of infection (J. Martín-García, unpubl. data). Further studies should therefore be carried out at different temperatures and with other techniques, such as fluorescence (
Oßwald
et al.
, 2014
) and molecular methods (
Bodles
et al.
, 2006
), to determine the damage caused beyond the visible symptoms.
Variation in the length (mm) of the necrotic
lesion (%) Pinus radiata D. Don, in plants inoculated with
isolates of Fusarium circinatum infected with mycoviruses
(FcMV1, FcMV2-2, and co-infection) relative to healthy
control plants inoculated with sterile distilled water. The
data were analyzed by the restricted maximum likelihood
(REML) for fixed effects.
Acknowledgements
The authors are grateful to the Instituto Agroforestal Mediterráneo (Polytechnic University of Valencia, Spain) for supplying the Fc104, Fc104v, Fc221 y Fc221w isolates of
F. circinatum
.
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