Effect of mycoviruses on the virulence of Fusarium circinatum and laccase activity

1 Effect of mycoviruses on the virulence of Fusarium circinatum 1 and laccase activity. 2 3 E. J. Muñoz-Adalia 1,2 *, J. A. Flores-Pacheco 1,2,3 , P. Martínez-Álvarez 1,2 , J. Martín-4 García 1,2 , M. Fernández 1,4 and J. J. Diez 1,2 . 5 6 7 1: Sustainable Forest Management Research Institute, University of Valladolid – INIA, 8 Avenida de Madrid 44, 34071 Palencia, Spain. 9 2: Department of Vegetal Production and Forest Resources, University of Valladolid. 10 Avenida de Madrid 44, 34071 Palencia, Spain. 11 3: Facultad de Recursos Naturales y Medio Ambiente, Bluefields Indian & Caribbean 12 University- BICU. Avenida Universitaria, Apartado postal N° 88 Bluefields, Nicaragua. 13 4: Department of Agroforestry Sciences, University of Valladolid. Avenida de Madrid 14 44, 34071 Palencia, Spain. 15 16 * Corresponding author: 17 E. Jordán Muñoz-Adalia. 18 Tel.: (34) 979108432. 19 Email: [email protected] / ejordanmu[email protected]m. 20 2 Abstract 21 22 Laccase enzymes (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) play a major role 23 in the degradation of phenolic compounds such as lignin. They are common in fungi and 24 have been suggested to participate in host colonization by pathogenic fungi. Putative 25 mycoviruses have recently been isolated from the causal agent of pine pitch canker 26 disease, Fusarium circinatum Nirenberg & O´Donell. In this study, the effects of single 27 and double mycoviral infections on laccase activity, growth rate and pathogenicity were 28 investigated in fourteen F. circinatum strains. Extracellular laccase activity was analyzed 29 by the Bavendamm test, image processing and a spectrophotometric method. Mycelial 30 growth, in vivo pathogenicity and seedling survival probability were also determined in 31 Monterrey pine (Pinus radiata D. Don) seedlings. The findings showed that (i) mycelial 32 growth of isolates from the same fungal population was homogeneous, (ii) the presence 33 of mycovirus appears to increase the virulence of fungal isolates, (iii) co-infection (with 34 two mycoviruses) caused cryptic effects in fungal isolates, and (iv) laccases embody a 35 possible auxiliary tool in fungal infection. The prospects for biocontrol, the adaptive role 36 of F. circinatum mycoviruses and the importance of laccase enzymes in host colonization 37 are discussed. 38 39 Keywords: Biocontrol, image analysis, multicopper oxidases, pine pitch canker disease, 40 ssRNA. 41 42 43 3 1. Introduction 44 45 Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) belong to the multicopper 46 oxidase group of enzymes and are specialized in catalyzing the oxidation of phenolic 47 substrates by reduction of O 2 to H 2 O. Laccases are common in eukaryotes, including 48 fungi, and have been widely studied in the phylum Ascomycota [1]. These enzymes 49 (molecular weight around 60-70 kDa) are usually extracellular and show a high degree 50 of specificity for degrading polyphenol substrates such as lignin [2]. They play an 51 essential role in nutrient turnover (mainly nitrogen and carbon) in nature, due to their 52 capacity to degrade lignocelluloses in forest soil and litter, and they are abundant in 53 saprophytic fungi [3]. Laccases may also play an important role in host colonization by 54 pathogenic fungi as they can damage host tissues, thus favouring fungal infection [4,5]. 55 Additionally, they have important applications in industry (e.g. textile and paper 56 industries) as well as in bioremediation and environmental biotechnology [6]. 57 58 The fungus Fusarium circinatum Nirenberg & O'Donnell is the causal agent of pine pitch 59 canker disease. This invasive necrotroph is considered the most important pathogen of 60 pine seedlings in several countries around the world and particularly affects conifers such 61 as Monterrey Pine (Pinus radiata D. Don) and Pseudotsuga menziesii (Mirb.) Franco 62 [7,8]. It can infect branches, stems, seeds, cones and roots in host trees of any age, 63 causing pre- and post-emergence damping-off in seedlings (mortality rates up to 90%) 64 and severe damage and reduced growth in adult trees [9]. Pine pitch canker fungus is 65 widespread throughout the world and has been reported in Mexico, USA, Haiti, South 66 Africa, Japan, Korea, Southern Europe and South America [10]. The pathogen spreads 67 via the movement of contaminated material (seeds, wood, nursery seedlings, etc.) as 68 well as via air- and soilborne spores and insect vectors [11] and via damage to trees 69 caused by storms or human activities [12]. The disease is expected to spread rapidly in 70 the future, and it has been estimated that approximately 10 million hectares of native 71 pine forest and plantations in the EU are potentially endangered [13]. 72 73 Several management measures and treatments for controlling F. circinatum have been 74 suggested: application of adaptive silviculture programmes [14], selection of particular 75 species for planting [8], treatment of seeds with hot water [15], addition of hydrogen 76 peroxide to irrigation water [16] and biocontrol techniques involving bacteria [17] or other 77 fungal species [18]. However, although some of these techniques are potentially useful, 78 new methods of biocontrol focused on field and nursery application are required. 79 80 4 Mycoviruses (viruses that infect fungi) are common in many fungal species, including 81 some plant pathogens [19]. Fifteen families of mycoviruses have been described: these 82 include single-strain RNA viruses which sequence serves as template for RNA-83 dependent RNA polymerase (RdRp) (ss(+)RNA), viruses that require the intervention of 84 RNA replicase to copy their genome into positive sense (ss(-)RNA) and also viruses with 85 double-strain RNA (dsRNA) and single-strain DNA (ssDNA) [20,21]. The effects of 86 mycoviruses on fungi vary from induction of a cryptic state to increase the capacity of 87 host to produce disease (hypervirulence). Although only a few mycoviruses reduce the 88 virulence of their host (hypovirulence), this kind of viruses is of particular interest for 89 biocontrol purposes [22]. One of the best known examples of virus-mediated 90 hypovirulence is that involving chestnut blight (causal agent Cryphonectria parasitica 91 (Murrill) M. E. Barr). Cryphonectria hypovirus 1 (CHV-1, Hypoviridae), which is one of 92 the four Hypovirus spp. that infects the fungus, has shown good results in biocontrol 93 treatment and has been shown to reduce fungal virulence (decreased mycelial growth 94 and sporulation rate) [23,24]. Other mycoviruses hosted by pathogenic fungi have also 95 been identified as promising organisms for biological control [25,26]. 96 97 Changes in laccase activity in fungi have been reported in relation to mycoviral infection 98 [27,28]. Laccase activity may also be altered in pathogenic fungi in the presence of 99 mycoviral infection, and reduced enzymatic activity may be associated with lower 100 virulence [29–31]. Three mycoviruses hosted in mitochondria that infect F. circinatum 101 have recently been identified as putative members of Narnaviridae (genus Mitovirus) and 102 designated Fusarium circinatum mitovirus 1, 2-1 and 2-2 (FcMV1, FcMV2-1 and FcMV2-103 2) [32]. Although little is known about the effects of these mycoviruses, any of them that 104 reduce laccase activity could potentially be used to develop a biocontrol technique to 105 treat pine pitch canker disease. 106 107 In this study, we hypothesized that F. circinatum isolates infected by mycoviruses would 108 show differences in laccase activity relative to isolates not infected by viruses. We also 109 expected to observe a positive correlation between laccase activity and host 110 pathogenicity. To our knowledge, this is the first study focusing on this topic in relation 111 to pine pitch canker disease. The objectives of this study were (i) to analyze the possible 112 effects of mycoviruses FcMV1 and FcMV2-2 on laccase activity in F. circinatum; (ii) to 113 investigate the variations in laccase activity, growth rates and infection development in 114 relation to mycovirus presence, and (iii) to evaluate the relationship between enzyme 115 activity and pathogenicity in Monterrey pine seedlings. 116 5 2. Material and Methods 117 118 2.1. Selection of isolates 119 120 Seven isolates of F. circinatum were obtained from two different locations in northern 121 Spain (Asturias and Cantabria) where wild-types of this fungus are commonly infected 122 by mycoviruses as previously reported [10]. Two monosporic cultures for each isolate 123 were selected, and the presence of mycoviruses was confirmed according to Álvarez et 124 al. [32]. The mating type (MAT) of each isolate was previously investigated [8] (Table 1). 125 Briefly, isolates FC104 and FC072 were free of mycovirus and isolates FC104v and 126 FC072v (i.e. of the same strains) were infected with FcMV1 (“v” indicates infection with 127 mycovirus). Isolate FC070v was also infected with FcMV1 and isolate FC070w was 128 infected with both FcMV1 and FcMV2-2 (“w” indicates co-infection). Isolates FC020, 129 FC035 and FC042 were free of mycovirus and FC020v, FC035v and FC042v were 130 infected with FcMV2-2. Finally, isolate FC221 was free of mycovirus and isolate FC221w 131 was co-infected with both mycoviruses. FcMV2-1 was not present in the evaluated 132 isolates. 133 134 2.2. Bavendamm test. 135 136 Seven samples of each isolate were cultured in Bavendamm medium to enable 137 estimation of the level of extracellular laccase activity. The fungal isolates were grown in 138 darkness at 25º C in specific media containing 0.50% w/v tannic acid, 1.50% w/v malt 139 extract and 2% w/v agarose. Tannic acid and malt-agarose solutions were prepared with 140 distilled water and autoclaved separately before being mixed together; the pH was 141 adjusted to 4.50 with NaOH 10M [31,33]. The global intensity of the enzymatic reaction 142 was evaluated after incubation for five days, and the change in color of the media (from 143 whitish to dark brown) was assessed according to the following qualitative scale: (-) non 144 appreciable reaction, (+) slight reaction or (++) intense reaction (Fig. 1). 145 146 2.3. Monitoring for mycelial growth. 147 148 In parallel to the Bavendamm test, photographs of the Petri dishes containing the fungal 149 isolates were taken every day for five days with a Canon EOS 550D camera (white backlit 150 screen as background and constant light). The photographs were processed using 151 ImageJ 1.48v [34] in order to quantify the area affected by enzymatic reaction (i.e. brown 152 area over whitish medium) [35,36]. The mean area affected by enzymatic reaction (S) 153 6 and mean growth of the isolate (G; calculated as the mean value of colony size increase 154 between two consecutive observations) were measured daily. 155 156 2.4. Laccase activity. 157 158 The F. circinatum isolates were cultured for one week in Bavendamm medium. Three 159 plugs (5x5 mm) comprising mycelia and medium were then removed from the edge of 160 each isolate and transferred to 1.50 ml tubes. Aliquots (1.50 ml) of twice-autoclaved 161 distilled water (4º C) were added to the plug samples to extract crude extracellular 162 laccase. After incubation for thirty minutes at room temperature, the tubes were 163 centrifuged for three minutes at 10 4 g and the supernatant was extracted. The laccase 164 activity was assayed after adding 0.80 ml of 2.50 mM 2,6-dimethoxyphenol (DMP, broad 165 spectrum enzyme substrate) to 0.20 ml of the crude laccase in 100 mM phosphate buffer 166 (pH 6.90) at 37º C [37]. The absorbance of samples was measured at 468 nm and 25º 167 C in a LAN OPTICS (2000-2100) spectrophotometer [38]. Absorbance was measured 168 immediately and five minutes later. Finally, the increase in absorbance was calculated 169 as an absolute value for the measurement period (ΔA 0-5 ). 170 171 2.5. In vivo pathogenicity. 172 173 To test in vivo the ability of each strain to cause disease (pathogenicity), the isolates 174 were inoculated into 405 one-year-old nursery seedlings of Monterrey pine (i.e. 27 175 replicate seedlings per isolate and 27 control seedlings). A small incision was made two 176 centimeters above the root collar and 10 μl of spore suspension (10 6 spores/ml of distilled 177 water) was inoculated into the wound. In control seedlings, an incision was made in the 178 same way, but distilled water only was inoculated into the wound. The wound was 179 covered with Parafilm ® for one week. The treated and control seedlings were held 180 separately in plant growth chambers at 25º C with a 16h photoperiod. The seedlings 181 were watered three times a week throughout the study period, with equal amounts of 182 distilled water. 183 184 After one week, the visual severity of symptoms in each plant were assessed every two 185 days during a period of 15 days, according to the following scale: 0 = healthy plant, 1 = 186 necrosis only at the point of inoculation and healthy foliage, 2 = necrosis >2 cm beyond 187 the point of inoculation, 3 = needles wilting and appreciable dieback and 4 = dead plant 188 [39] (Fig. 1). Finally, the area under the disease progress curve (AUDPC) was calculated 189 as the sum of the area of the corresponding trapezoids as previously described [8]. 190 7 191 2.6. Statistical analysis. 192 193 All analyses were performed with R software [40]. The Kruskal-Wallis rank sum tests 194 were carried out with the “Agricolae” package [41] to analyze the variation in S, G, ΔA 0-5 195 and AUDPC values according to two different factors: isolate (14 strains, Table 1) and 196 mycovirus presence (evaluated as follows: not infected (Ø); infected with FcMV1 or 197 FcMV2-2; and co-infected with both mycoviruses). Dunn´s test [42] was applied for post-198 hoc analysis of data, with “DescTools” package [43]. The Pearson's product-moment 199 correlation [44] was also calculated for (a) G and ΔA 0-5 , (b) mean values of AUDPC and 200 ΔA 0-5 for each isolate, and (c) the G and S variables. Survival analysis based on the non-201 parametric Kaplan-Meier estimator [45] was carried out with “Survival” package [46]. 202 Survival curves were created with the “Survfit” function and the differences between the 203 curves were tested with the “Survdiff” function. 204 205 <<Insert Figure 1 around here>> 206 8 3. Results 207 208 3.1. Bavendamm test and mycelial growth. 209 210 All isolates showed an intense response in the Bavendamm test (Table 1). The mean 211 value of S was 491.27 ± 27.85 mm 2 (standard error). The Kruskal-Wallis rank sum test 212 revealed significant differences in S between isolates (Х 2 = 37.45; d.f.= 13; P= <0.01) but 213 not in relation to mycovirus presence (Х 2 = 0.94; d.f.= 3; P= 0.81). Isolate FC104 yielded 214 the highest value of S (mean value 724.22 ± 31.21 mm 2 ), which was significantly different 215 from the values yielded by other isolates, including FC104v (P= <0.01). FC042 and 216 FC070v resulted in the lowest S values, without significant differences between them 217 (P= 0.47). The S values produced by these isolates and the non-infected pairs (FC042 218 and FC070) were not significantly different (P= 0.15; P= 0.21, respectively) (Fig. 2). The 219 G and S variables were closely correlated (t= 19.04; d.f.= 96; P= <0.01; ρ= 0.88). 220 221 The isolates grew quickly, and the mean G value was 227.28 ± 16.83 mm 2 /day. Growth 222 did not vary significantly in relation to mycovirus presence (Х 2 = 2.13; d.f.= 3; P= 0.54) 223 and it also did not differ significantly between isolates (Х 2 = 22.27; d.f.= 13; P= 0.05). 224 225 <<Insert Table 1 and Figure 2 around here>> 226 227 3.2. Laccase activity. 228 229 Laccase activity (expressed as ΔA 0-5 ) differed significantly in relation to the isolate (Х 2 = 230 22.54; d.f.= 13; P= 0.04), whereas the presence of the mycovirus did not have a 231 significant effect (Х 2 = 1.92; d.f.= 3; P= 0.58). Of the fungal isolates infected with 232 mycovirus, FC042v produced the greatest increase in the absorbance, which was 233 significantly different from that produced by the same isolate not infected with the 234 mycovirus, which yielded the lowest absorbance increase (FC042, P= <0.01). Likewise, 235 ΔA 0-5 also differed significantly between FC104 and FC104v (P= 0.01) (Fig. 3) but the 236 correlation between G and ΔA 0-5 was not significantly different (t= -0.88; d.f.= 96; P= 0.37; 237 ρ= -0.09). 238 239 <<Insert Figure 3 around here>> 240 241 242 3.3. Pathogenicity in vivo. 243 9 244 The values of AUDPC obtained in relation to the different treatments varied significantly 245 depending on the isolate (Х 2 = 98.90; d.f.= 14; P= <0.01). The highest AUDPC value was 246 obtained for FC072v and it was significantly different from that obtained for its pair FC072 247 (P= 0.02). The lowest value was obtained for seedlings infected with FC042 (mean value 248 36.92 ± 2.17) and was significant different from the values corresponding to the other 249 isolates (P= <0.03, in all cases) (Fig. 4). 250 251 <<Insert Figure 4 around here>> 252 253 The AUDPC also varied significantly in regard to viral infection (Х 2 = 25.75; d.f.= 3; P= 254 <0.01). The value was higher in all plants infected by F. circinatum isolates than in control 255 seedlings, as expected (<0.01, in all cases). The AUDPC values were higher in FcMV1-256 infected fungi than in non-infected (P= <0.01) and co-infected isolates (P= 0.02), but 257 there were no significant differences between FcMV2-2 infected isolates (P= 0.11). 258 There were no significant differences between co-infected isolates and either isolates 259 infected with FcMV2-2 only (P= 0.16) or non-infected isolates (P= 0.40) (Fig. 5). The 260 correlation between AUDPC and ΔA 0-5 as average values for each isolate were almost 261 statistically significant (t= 2.13; d.f.= 13; P= 0.05; ρ= 0.50). 262 263 <<Insert Figure 5 around here>> 264 265 Survival analysis revealed significant differences between treatments (Х 2 = 94.50; d.f.= 266 4; P= <0.01) (Fig. 6). The survival probability of seedlings was significantly lower in plants 267 inoculated with isolates infected with FcMV1 than in the virus-free isolates (Х 2 = 11.10; 268 d.f.= 1; P= <0.01). FcMV2-2 presence in fungi did not produce any differences in plant 269 host survival relative to non-infected isolates (Х 2 = 3.30; d.f.= 1; P= 0.06). No differences 270 were found in seedlings survival probability between isolates infected with FcMV1 or 271 FcMV2-2 (Х 2 = 1.50; d.f.= 1; P= 0.22). 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Data and results of tests of Fusarium circinatum isolates (seven isolates, two 567 monosporic cultures/isolate): origin; host (Pp: Pinus pinaster Aiton, Pr: Pinus radiata); 568 mating-type (MAT); mycovirus presence (FcMV1/FcMV2-2); intensity of Bavendamm 569 test reaction (B.t.; qualitative scale: -, +, ++); area affected by enzymatic reaction (S); 570 mycelial growth (G); increase of absorbance in five minutes (ΔA 0-5 ) and area under the 571 disease progress curve (AUDPC). Mean values and standard error (SE) are shown. (*) 572 Source of data: [47].573 21 Isolate Origin Host MAT FcMV1 FcMV2-2 B.t. S (mm 2 ) ± SE G (mm 2 /day) ± SE ΔA 0-5 ± SE AUDPC ± SE FC104v Asturias* Pp* 1*  - (++) 524.98 ± 55.15 226.21 ± 35.50 0.09 ± 0.04 44.33 ± 1.75 FC072v Cantabria Pr 2  - (++) 519.32 ± 44.11 252.71 ± 22.62 0.08 ± 0.02 43.35 ± 1.52 FC070v Cantabria Pr 2  - (++) 387.36 ± 45.70 174.91 ± 27.79 0.24 ± 0.06 46.46 ± 1.44 FC070w Cantabria Pr 2   (++) 443.03 ± 51.04 224.61 ± 28.30 0.14 ± 0.04 45.40 ± 1.34 FC221w Cantabria* Pr* 2*   (++) 542.83 ± 36.29 229.24 ± 26.57 0.15 ± 0.03 43.37 ± 1.36 FC020v Cantabria Pr 2 -  (++) 480.60 ± 38.29 220.02 ± 27.82 0.10 ± 0.03 46.29 ± 1.56 FC035v Cantabria Pr 2 -  (++) 503.56 ± 43.11 229.89 ± 21.44 0.13 ± 0.04 47.12 ± 1.22 FC042v Cantabria Pr 2 -  (++) 447.40 ± 34.71 229.67 ± 22.57 0.32 ± 0.07 49.03 ± 1.63 FC104 Asturias* Pp* 1* - - (++) 724.22 ± 31.21 332.74 ± 13.01 0.23 ± 0.04 45.14 ± 1.31 FC072 Cantabria Pr 2 - - (++) 504.00 ± 47.13 199.99 ± 34.72 0.10 ± 0.05 41.79 ± 2.14 FC221 Cantabria* Pr* 2* - - (++) 515.79 ± 46.29 233.29 ± 25.94 0.44 ± 0.15 45.24 ± 1.54 FC020 Cantabria Pr 2 - - (++) 482.57 ± 35.56 231.64 ± 22.50 0.08 ± 0.01 36.92 ± 2.17 FC035 Cantabria Pr 2 - - (++) 417.13 ± 27.38 202.97 ± 14.82 0.14 ± 0.07 45.11 ± 1.32 FC042 Cantabria Pr 2 - - (++) 384.91 ± 15.46 203.73 ± 12.62 0.07 ± 0.02 44.33 ± 1.45 22 Figures Fig. 1. Scheme of the study. A: Bavendamm test progress at four different moments: 24 h (a), 48 h (b), 72 h (c) and 96 h (d) after culture (isolate shown: Fc072). B: Control Pinus radiata seedlings on the 13 th day of pathogenicity test. C: Pinus radiata seedlings inoculated with Fc072v (foreground) and Fc072 (background) on the 13 th day of pathogenicity test. D: Detail of resin surrounding the point of inoculation. E: Detail of dead seedling showing the symptomatology of pine pitch canker damping-off. 23 Fig. 2. Area affected by enzymatic reaction during the five days of the assay (S) for each fungal isolate. Small letters (a–e) denote significant differences (Dunn´s test, P= <0.05). (Ø): mycovirus-free isolates. Comparisons between pairs of isolates are indicated by color of plot and roman numbers (I-VII). Median values and standard error are shown. Fig. 3. Extracellular laccase activity (ΔA 0-5 ) in the different isolates. Small letters (a–c) denote significant differences (Dunn´s test, P= <0.05). (Ø): virus-free isolates. Comparisons between pairs of isolates are indicated by color of plot and roman numbers (I-VII). Median values and standard error are shown. 24 Fig. 4. Area under the disease progress curve (AUDPC) for the different fungal isolates. Small letters (a–d) denote significant differences (Dunn´s test, P= <0.05). (Ø): virus-free isolates. Comparisons between pairs of isolates are indicated by color of plot and roman numbers (I-VII). Median values and standard error are shown. Fig. 5. Area under the disease progress curve (AUDPC) in relation to mycovirus presence. Small letters (a–c) denote significant differences (Dunn´s test, P= <0.05). (Ø): virus-free isolates. Median values and standard error are shown. 25 Fig. 6. Plot of survival probability determined using the Kaplan-Meier estimate of the survival function for Monterrey pine (Pinus radiata) seedlings infected with Fusarium circinatum in relation to mycovirus presence. (Ø): mycovirus-free isolates.