SHORT COMMUNICATION

 

Effect of mycoviruses on growth, spore germination and pathogenicity of the fungus Fusarium circinatumt

 

Juan Asdrúbal Flores-Pacheco

University of Valladolid - INIA, Sustainable Forest Management Research Institute, Avda. de Madrid 44, 34071 Palencia, Spain.

ETSIIAA, Dept. of Plant Production and Forestry Resources, Avda. de Madrid 57, 34004 Palencia, Spain.

Facultad de Recursos Naturales y Medio Ambiente, Bluefields Indian & Caribbean University- BICU 88, Avda. Universitaria, Bluefields, Nicaragua.

Emigdio Jordan Muñoz-Adalia

University of Valladolid - INIA, Sustainable Forest Management Research Institute, Avda. de Madrid 44, 34071 Palencia, Spain.

ETSIIAA, Dept. of Plant Production and Forestry Resources, Avda. de Madrid 57, 34004 Palencia, Spain.

Pablo Martínez-Álvarez

University of Valladolid - INIA, Sustainable Forest Management Research Institute, Avda. de Madrid 44, 34071 Palencia, Spain.

ETSIIAA, Dept. of Plant Production and Forestry Resources, Avda. de Madrid 57, 34004 Palencia, Spain.

Valentín Pando

University of Valladolid - INIA, Sustainable Forest Management Research Institute, Avda. de Madrid 44, 34071 Palencia, Spain.

University of Valladolid, Statistics and Operations Research Dept., Avda. de Madrid 57, 34004 Palencia, Spain.

Julio J. Diez-Casero

University of Valladolid - INIA, Sustainable Forest Management Research Institute, Avda. de Madrid 44, 34071 Palencia, Spain.

ETSIIAA, Dept. of Plant Production and Forestry Resources, Avda. de Madrid 57, 34004 Palencia, Spain.

Jorge Martín-García

University of Valladolid - INIA, Sustainable Forest Management Research Institute, Avda. de Madrid 44, 34071 Palencia, Spain.

University of Aveiro, CESAM, Dept. of Biology, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal

 

Abstract

Aim of the 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.

Additional Keywords: pine pitch canker; hypovirulence; biological control; forest pathology.

Abbreviations used: FcMV (Fusarium circinatum Mitovirus); PDB (potato dextrose broth); PPC (pine pitch canker).

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.

Citation: Flores-Pacheco, J. A.; Muñoz-Adalia, E. J.; Martínez-álvarez, P.; Pando, V.; Díez-Casero, J. J.; Martín-García, J. (2017). Short communication: Effect of mycoviruses on growth, spore germination and pathogenicity of the fungus Fusarium circinatum. Forest Systems, Volume 26, Issue 3, eSC07. https://doi.org/10.5424/fs/2017263-11060

Received: 16 Jan 2017. Accepted: 04 Dec 2017.

Copyright © 2017 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License.

Funding: 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.

Correspondence should be addressed to Juan Asdrúbal Flores-Pacheco: juan18asdrubal@gmail.com; japacheco@pvs.uva.es


 

CONTENTS

Abstract

Introduction

Material and methods

Results and discussion

Acknowledgements

References

IntroductionTop

Fungal isolates

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 methodsTop

Fungal 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.

Table 1. 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

Mycelial growth of F. circinatum isolates was evaluated on Petri dishes containing PDAS medium (potato dextrose agar and 0.5 mg/L streptomycin). A 4-mm mycelial plug of the pathogen was placed in the centre of the plate. The plates were sealed with Parafilm© and kept in darkness at 25°C for seven days (Leslie & Summerell, 2006). Seven replicates of each isolate were used in the assays. The growth of the colonies (along 2 perpendicular axes) was evaluated daily, and the total area of growth was calculated by the following equation:

Area = ∏/4 (D1 х D2),

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 21° C with a 16-h photoperiod, following a completely randomized design.

Inoculations were carried out by the stem inoculation technique (Martínez-álvarez et al., 2016). U-shaped wounds were cut into the bark of the seedlings, at 5-7 cm above ground, with a sterile scalpel (Correll et al., 1991). The conidia suspension was obtained from F. circinatum isolates cultured for seven days in PDB at 25°C and darkness. Aliquots (100 μL) of spore suspension (adjusted to 106 conidia/mL using a hemocytometer) were inoculated into the wound, which was covered with Parafilm© to prevent drying (Martínez-álvarez et al., 2014a). Twenty-seven seedlings were inoculated for each isolate tested. Control seedlings (n=27) were prepared in the same way with sterilized distilled water instead of spore suspensions.

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 discussionTop

Effect 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.

Figure 1. 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 (Oβwald et al., 2014) and molecular methods (Bodles et al., 2006), to determine the damage caused beyond the visible symptoms.

Table 2. 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.

AcknowledgementsTop

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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