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11 6 The cacao pathogen Moniliophthora roreri (Marasmiaceae) produces rhexolytic thallic
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13 7 conidia and their size is influenced by nuclear condition
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16 9 Jorge R. Díaz-Valderrama*, M. Catherine Aime
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18 11 Department of Botany and Plant Pathology, Purdue University,
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20 12 915 West State St., West Lafayette, Indiana, 47907, USA
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22 *
23 14 Corresponding author:
24 15 Jorge R. Díaz-Valderrama
25 16 Fax: +1 (765) 494-0363
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27 17 Phone: +1 (765) 494-7791
28 18 jdiazval@purdue.edu
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63 © 2016. This manuscript version is made available under the Elsevier user license
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4 23 Abstract
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7 25 Moniliophthora roreri, the causal agent of frosty pod rot of cacao, is a member of the
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9 26 mushroom-forming family Marasmiaceae (Marasmiineae, Agaricales, Basidiomycota). Yet, M.
10 27 roreri has never been observed to produce a mushroom fruiting body, but rather produces
11 28 billions of spores on the surface of infected pods. The question of whether these spores are
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13 29 produced via meiosis or mitosis has been the subject of some speculation. However, numerous
14 30 molecular-based studies have been unable to support a hypothesis of sexual recombination for
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16 31 this fungus. We re-examined sporogenesis and the nuclear condition of hyphae and spores in M.
17 32 roreri via nuclear staining and spore germination studies. Conidia are produced asexually in a
18 33 thallic and rhexolytic manner as is true for other Marasmiineae species such as M. perniciosa,
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20 34 Flammulina velutipes and Marasmius puerariae. We also found that hyphal cells as well as
21 35 spores harbor one or two nuclei, rarely three, that conidium size is influenced by number of
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23 36 nuclei within, and that individual isolates produced consistently and significantly different
24 37 proportions of binucleate and mononucleate spores regardless of varietal group.
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27 39
28 40 Keywords
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31 42 Anamorph, Dikaryon, Fungal mating, Spore ontogeny, Tropical plant diseases
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4 44 1. Introduction
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7 45 Frosty pod rot of cacao (FPR), caused by Moniliophthora roreri H.C. Evans, Stalpers, Samson &
8 46 Benny, is one of the primary culprits of cacao yield losses in Latin America and constitutes a
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10 47 huge threat for the world cacao production due to its invasive behavior (Phillips-Mora and
11 48 Wilkinson 2007). Before the 1950’s the fungus was confined to North-Western South America
12 49 (Ecuador, Colombia and Western Venezuela) but in the last decades it has expanded its
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14 50 geographical range to almost the entire Latin American cacao-growing region, from Bolivia to
15 51 Mexico (Griffith et al. 2003; Aime and Phillips-Mora 2005; Phillips-Mora et al. 2006a, b, 2007,
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17 52 2015). Currently, M. roreri constitutes a huge threat to Brazil, the major cacao-producing
18 53 country in South America and the only one in which FPR is still not present (Phillips-Mora and
19 54 Wilkinson 2007).
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21 55 The taxonomic position of of M. roreri has been controversial throughout the years
22 56 mainly due to its lack of production of an observable sexual stage. Initially, it was described as
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24 57 an anamorphic ascomycetous fungus, Monilia roreri, due to similar symptomathology and
25 58 morphology to the ascomycetous plant pathogen Monilinia fructigena (Ciferri and Parodi 1933).
26 59 However, ultrastructural obervations of its dolipore septum led its transfer to the Basidiomycota
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28 60 and the creation of a new genus name, Moniliophthora, to accommodate it although its specific
29 61 taxonomic placement within the phyllum remained uncertain (Evans et al. 1978). More recent
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31 62 molecular analysis of rDNA loci and other phylogenetic markers finally revealed that M. roreri
32 63 belongs to the Marasmiaceae (Marasmiineae, Agaricales) (Aime and Phillips-Mora 2005). Two
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64 varieties of M. roreri have been described, differentiated primarily by spore size and host (Evans
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35 65 et al. 2003b). Moniliophthora roreri var. roreri produces smaller spores (5.5–12.5 × 5.0–10.0
36 66 µm) and infects a range of Theobroma and Herrania species, whereas M. roreri var. gileri
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38 67 produces larger spores (8.0–16.0 × 5.5–11.0 µm) and is restricted to T. gileri as a host species
39 68 (Evans et al. 2003b).
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69 The spore production capacity of M. roreri is enormous and can exceed seven billion
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42 70 spores per infected pod (Campuzano 1976). Historically, these spore have been characterized as
43 71 conidia, but recent cytological analyses have reinterpreted them as meiospores produced in a
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45 72 modified basidium, although neither evidence of karyogamy nor production of mating
46 73 compatible haploid spores has been found (Evans et al. 2003a, b, 2013). Moreover, independent
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74 population studies have not found evidence for sexual recombination in this fungus (Grisales
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49 75 Ortega and Kafuri 2007; Phillips-Mora et al. 2007), which appears to spread by strictly clonal
50 76 propagation (Díaz-Valderrama and Aime 2016).
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52 77 Members of the Agaricales are widely known for the production of sexual spores or
53 78 basidiospores that are the product of meiosis and produced externally on the hallmark basidia for
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79 which the phylum is named. However, the presence of asexual spores or conidia in this group is
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56 80 also widespread and most of the families within it have at least one species known to sporulate
57 81 asexually in culture and/or in nature (Ingold 1980; Walther et al. 2005), which can occur on
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59 82 either monokaryotic or dikaryotic hyphae (Kaufert 1935; Delgado and Cook 1976; Ingold 1980;
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4 83 Kertesz-Chaloupková et al. 1998; Walther and Weiß 2008; Badalyan et al. 2011). Overall, there
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6 84 are two main modes of conidiogenesis in the Agaricales: blastic, in which conidia are produced
7 85 from specialized conidiogenous cells before they are delimited by septa; and thallic, when
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9 86 conidia are produced by fragmentation, or secession, of hyphal cells after they have been
10 87 delimited by septa (Moore et al. 2011). Blastic conidiogenesis is uncommon in Agaricales, and
11 88 has been observed in only a few species: Baeospora myosura (Cyphellaceae), Gymnopilus
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13 89 junonius (Gymnopileae) and some members of Hohenbuehelia (Pleurotaceae) (Walther et al.
14 90 2005). Thallic conidiogenesis, on the other hand, is predominant in most of the families of the
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16 91 Agaricales (Walther et al. 2005) and is further characterized by two modes of conidia secession:
17 92 schizolytic, wherein the septa itself splits to form the two daughter conidia; and rhexolytic, when
18 93 plasma in hyphal cells contracts creating plasma-free compartments that break to form the
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20 94 conidia (Cole and Samson 1979; Walther et al. 2005). Most of the members in the Marasmiineae
21 95 suborder, to which M. roreri belongs (Dentinger et al. 2015), have not been observed to develop
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23 96 asexual spores (Walther et al. 2005), with the exception of Flammulina velutipes and other
24 97 Flammulina species (Physalacriaceae), Moniliophthora perniciosa, Marasmius puerariae
25 98 (Marasmiaceae) and Mycena citricolor (Mycenaceae), which display rhexolytic thallic
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27 99 conidiation (Delgado and Cook 1976; Ingold 1980; Petersen 1995; Petersen et al. 1999; Redhead
28 100 et al. 2000; Kirschner et al. 2013; Dentinger et al. 2015).
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30 101 Moniliophthora roreri is a highly unusual fungus that severely affects cacao production
31 102 in Latin America. However, more than for other agarics, little is known of its most basic biology.
32 103 Crucially, accurate understanding of how this fungus reproduces is essential to developing
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34 104 effective control measures. Hence, the objective of this study was to determine how spores in M.
35 105 roreri are produced.
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37 106
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39 107 2. Materials and Methods
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108 2.1. Single spore isolations
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44 110 For this study we used single and non-single-spored cultures of M. roreri obtained from diseased
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46 111 pods from Ecuador, Mexico and Peru (Table 1). To perform single-spore isolations (SSI), we
47 112 harvested spores using a method developed for Coprinopsis cinerea transformation (Dörnte and
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113 Kües 2012). Cultures were grown on potato dextrose agar (PDA) media at room temperature for
50 114 3 to 4 wk prior to spore harvest. Culture plates were flooded with sterile water and spores were
51 115 scraped from the surface with spatula and then filtered through sterilized glass wool and
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53 116 collected into a Sterilin tube. Filtrate was then serially diluted (1/25-, 1/625- and sometimes
54 117 1/15625-fold dilutions of an initial ca. 20 ml solution). Approximately 1 ml of each dilution was
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118 plated on petri plates containing PDA (3.9% w/v) + charcoal (1% w/v) medium. Charcoal was
57 119 used because it has been suggested to stimulate spore germination in other Agaricales species
58 120 (Fries 1978). After plating, the plates were incubated in the dark at room temperature (ca. 25 °C)
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4 121 and monitored daily for the presence of germination. Individual germinating spores that were not
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6 122 touching each other were transferred to a new PDA plate as soon as germination was visible
7 123 (around 14 d after plating).
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9 124
10 125 2.2. Conidiogenesis, nuclear condition and spore size
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13 127 Conidiogenesis was determined by light microscopy observations of hyphae, conidiophores and
14 128 mature conidia of isolates using the key for identification of Agaricales anamorphs based on
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16 129 conidia formation (Walther et al. 2005). To determine the nuclear condition, hyphae and spores
17 130 from 2- to 4-wk-old cultures were mounted and stained with VECTASHIELD® mounting
18 131 medium with DAPI (4,6 diamidino-2-phenylindole; Kapuscinski 1995) (VECTOR Laboratories,
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20 132 Burlingame, CA) and examined under bright and fluorescent light microscopy. To look at the
21 133 behavior of nuclei during spore germination, spores from isolate CBS 138634 were plated on
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23 134 PDA and allowed to germinate for 24 h, after which the plates were flooded with sterile water
24 135 and the conidia in solution were stained and examined as previously described.
25 136 Comparison of mono and binucleate spores was performed by using two variables:
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27 137 average diameter and area. Two perpendicular measures of the diameter were taken from the
28 138 spores and the average diameter was calculated using software ImageJ1.46; from these two
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30 139 diameters, the area of the conidia plane observed under the microscope was calculated with the
31 140 formula: Spores from four single-spored cultures were used
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33 141 for this comparison: DIS 371, CBS 138632, CBS 138631, and CBS 138635. Fifty-nine, fifty-
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142 eight, three-hundred and sixty-two, and one-hundred and seven spores were evaluated per
36 143 isolate, respectively. Analysis of variance (ANOVA) and Tukey- Honest Significant Difference
37 144 (HSD) tests were carried out separately per isolate with software RStudio v0.97.551. Finally, the
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39 145 proportions of binucleate and mononucleate spores produced by isolates were recorded and
40 146 analyzed with a Marascuilo test to determine significant differences.
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43 148 3. Results
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45 149 3.1. Conidiogenesis, nuclear condition and spore size
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48 151 Spores of M. roreri from single-spored and non-single-spored cultures are produced asexually in
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152 a manner indistinguishable from that of conidiogenesis of F. velutipes (Marasmiineae) according
51 153 to the identification key to the conidia-forming anamorphs of Agaricales (Walther et al. 2005).
52 154 Conidia do not develop from clamp connections or pseudo clamps and their formation is strictly
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54 155 thallic, produced directly by the fragmentation of the conidiogenous hyphal cells, with rhexolytic
55 156 secession, which includes plasma contraction and formation of plasma-free compartments (Figs.
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157 1, 2). Conidiophores are not differentiated in a sterile stipe or a fertile apical part and
58 158 conidiogenous hyphae are not coiled, features of conidiogenesis in other families and suborders
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4 159 of the Agaricales (Figs. 1, 2). In addition, conidiogenous cells in hyphae swell during maturation
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6 160 producing globose conidia (Figs. 1B, E, 3).
7 161 The DAPI staining of hyphae from single-spored as well as non-single-spored cultures
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9 162 showed the nuclear condition of these are variable but most found to contain either one or two
10 163 nuclei (Figs. 1, 2). The number of nuclei per conidium is also variable, with the vast majority
11 164 containing one or two nuclei, and rarely three (Fig. 3). Binucleate spores (5.7–18.6 × 4.6–13.5
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13 165 µm) are significantly larger in average diameter and area than mononucleate spores (5.4–18.4 ×
14 166 3.7–9.4 µm), with no differences found between varieties (Fig. 4; Table 2). The average diameter
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16 167 and area of binucleate spores are 9.1 µm and 64.1 µm2, respectively, whereas the average
17 168 diameter and area of mononucleate spores are 7.3 µm and 41.9 µm2, respectively (Fig. 4). One
18 169 interesting finding was that although the sizes of the spores produced by each isolate was
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20 170 consistent between isolates, some isolates were found to produce significantly different
21 171 proportions of binucleate and mononucleate spores (p < 0.001), which seemed to be a
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23 172 characteristic of isolate rather than variety. Isolates DIS 371 (var. gileri) and CBS 138635 (var.
24 173 roreri) produce the largest proportion of binucleate spores, while isolate CBS 138631 (var.
25 174 roreri) produced the smallest proportion (Table 2). Finally, germination tests shows both
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27 175 binucleate and mononucleate spores are able to germinate, producing monokaryotic hyphae (Fig.
28 176 5). No evidence of meiosis during conidiation or germination was found (Fig. 5).
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31 178 4. Discussion
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179 Our results support a hypothesis of asexual production of spores in M. roreri. Conidiogenesis in
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35 180 the fungus is thallic, and conidia are produced in a rhexolytic manner (Fig. 6) similar to that of
36 181 the other Marasmiineae species for which conidiation has been studied (F. velutipes, M.
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38 182 perniciosa and M. puerariae) (Delgado and Cook 1976; Ingold 1980; Walther et al. 2005;
39 183 Kirschner et al. 2013). The hallmarks of rhexolytic conidiogenesis—swelling of conidia during
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184 maturation, plasma contraction in the conidiogenous hyphae, and thallic secession—are present
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42 185 in M. roreri, and it even can be found in prior illustrations of the fungus (e.g., Ciferri and Parodi
43 186 1933; Suárez 1971; Evans et al. 1978, 2003b; Bailey et al. 2013; Figs. 1, 5).
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45 187 Hyphal cells and conidia in M. roreri contain variable numbers of nuclei (Figs. 1–3).
46 188 Clamp connections were never observed, as already noted (Ciferri and Parodi 1933; Evans et al.
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189 1978), although its sister species, M. perniciosa, is known to produce clamps on its
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49 190 conidiogenous hyphae (Delgado and Cook 1976). Vegetative hyphae cells of M. roreri were
50 191 observed to contain mostly one or two nuclei (Griffith et al. 2003; Figs. 1, 2) which appears to be
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52 192 a characteristic of homokaryotic hyphae of other agarics such as Coprinopsis cinerea (Kües et al.
53 193 2002). Because number of nuclei varies among the cells of a single hypha, subsequent
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194 conidiogenesis ensures that varying numbers of nuclei are partitioned into the conidia (Figs. 3,
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56 195 6). In contrast, conidia from the sister species, M. perniciosa, are exclusively binucleate because
57 196 they are produced on dikaryotic clamped hyphae that are all regularly binucleate (Delgado and
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59 197 Cook 1976). Conidia from F. velutipes can come from either monokaryotic or dikaryotic hyphae
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4 198 but the cultures they produce when seeded in new culture plates are always monokaryotic (Kemp
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6 199 1980). In the case of M. roreri, both uninucleate and binucleate conidia germinate and produce
7 200 monokaryotic hyphae (Fig. 5), supported also by the fact that all the single-spored cultures are
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9 201 monokaryotic, assuming isolated spores were uninucleate or binucleate. A dikaryotic stage in M.
10 202 roreri, where the presence of two mating-type compatible nuclei occurs per single cell, has never
11 203 been observed which reconfirms that conidia from the same conidiogenous hyphal chain have
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13 204 the same mating incompatibility type (Aschan 1952); otherwise we would expect to have a
14 205 dikaryotic stage in M. roreri, characterized by the production of exclusively binucleate (or,
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16 206 hypothetically, diploid) hyphae. The hemibiotrophic lifestyle of M. roreri has recently been
17 207 deciphered, which might involve the production of dikaryotic hyphae during the necrotrophic
18 208 phase of infection (Bailey et al. 2013; Meinhardt et al. 2014). However, despite this possibility,
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20 209 no evidence for sexual recombination, has been found in M. roreri, which appears to be
21 210 reproducing clonally (Díaz-Valderrama and Aime 2016). All these sources of evidence support
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23 211 the hypothesis that this fungus is likely an exclusively anamorphic fungus as originally described
24 212 (Ciferri and Parodi 1933).
25 213 Some studies have shown that the dimension of a mature conidium can be related to the
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27 214 DNA content and number of nuclei in the conidial cell (Leaich and Papa 1975; Phillips et al.
28 215 1987), which supports our findings that the binucleate conidia were significantly larger than
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30 216 uninucleate conidia in average diameter and area (Fig. 4). Interestingly, we found that the
31 217 proportion of binucleate versus mononucleate conidia produced by isolates was a consistent
32 218 feature of the isolate, and that these could vary significantly between isolates (Table 2); for
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34 219 example isolate CBS 138631 (M. roreri var. roreri) produces significantly less binucleate spores
35 220 than the other isolates. Furthermore, we found no significant differences in spore sizes between
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37 221 the isolates from different varieties (Fig. 4) despite the use of conidial size as the defining
38 222 morphological character for diagnosing between these varieties (Evans et al. 2003b). Together,
39 223 this suggests that differences previously found in conidia size between different isolates of the
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41 224 fungus from different hosts (Evans et al. 2003b) are likely due to interstrain variability in nuclei
42 225 proportions and not evidence of intraspecific varieties.
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44 226
45 227 Disclosure
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48 229 Authors declare no conflict of interest.
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51 231
52 232 Acknowledgments
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55 234 We thank Drs. Wilbert Phillips-Mora (Tropical Agricultural Research and Higher Education
56 235 Center CATIE, Costa Rica), Enrique Arévalo-Gardini (Institute of Tropical Crops, Peru),
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58 236 Francisco Posada (USDA-ARS) and Carmen Suárez (Technical State University of Quevedo,
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4 237 Ecuador) for providing the original isolates that were used in this study. We also thank Dr. Chris
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6 238 Gilpin and Laurie Mueller from the Purdue Life Science Microscopy Facility for facilitating
7 239 bright-light and fluorescent microscopes. We acknowledge the Purdue University Department of
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9 240 Botany and Plant Pathology for supporting this work.
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4 241 REFERENCES
5
6
7
8 242 Aime MC, Phillips-Mora W, 2005. The causal agents of witches’ broom and frosty pod rot of
9 243 cacao (chocolate, Theobroma cacao) form a new lineage of Marasmiaceae. Mycologia
10
11
244 97: 1012–1022; http://dx.doi.org/10.3852/mycologia.97.5.1012.
12 245 Aschan K, 1952. Studies on dediploidization mycelia of the basidiomycete Collybia velutipes.
13 246 Svensk Botanisk Tidskrif 46: 366–392.
14 247 Badalyan SM, Navarro-González M, Kües U, 2011. Taxonomic significance of anamorphic
15 248 characteristics in the life cycle of coprinoid mushrooms. Proceedings of the 7th
16
17 249 International Conference on Mushroom Biology and Mushroom Products (ICMBMP7),
18 250 Arcachon, France, pp 140–154.
19 251 Bailey BA, Crozier J, Sicher RC, Strem MD, Melnick R, Carazzolle MF, Costa GGL, Pereira
20 252 GAG, Zhang D, Maximova S, Guiltinan M, Meinhardt L, 2013. Dynamic changes in pod
21
253 and fungal physiology associated with the shift from biotrophy to necrotrophy during the
22
23 254 infection of Theobroma cacao by Moniliophthora roreri. Physiological and Molecular
24 255 Plant Pathology 81: 84–96; doi:10.1016/j.pmpp.2012.11.005.
25 256 Campuzano H, 1976. Fluctuación de poblaciones de esporas de Monilia roreri Cif. & Par. y
26 257 viabilidad durante un ciclo completo de afección. Noticias Fitopatológicas (Colombia) 5:
27
28
258 107.
29 259 Ciferri R, Parodi E, 1933. Descrizione del fungo che causa la Moniliasi del cacao.
30 260 Phytopathologische Zeitschrift 6: 539–542.
31 261 Cole GT, Samson RA, 1979. Patterns of development in conidial fungi. Pitman, London.
32 262 Delgado JC, Cook AA, 1976. Arthroconidia formation in cultures of Marasmius perniciosus.
33
34 263 Phytopathology 66: 717–718.
35 264 Dentinger BTM, Gaya E, Brien HO, Suz LM, Lachlan R, Díaz-Valderrama JR, Koch RA, Aime
36 265 MC, 2015. Tales from the crypt: genome mining from fungarium specimens improves
37 266 resolution of the mushroom tree of life. Biological Journal of the Linnean Society 117:
38
39
267 11–32; doi:10.1111/bij.12553.
40 268 Díaz-Valderrama JR, Aime MC, 2016. The cacao pathogen Moniliophthora roreri
41 269 (Marasmiaceae) possesses biallelic A and B mating loci but reproduces clonally. Heredity
42 270 In press.
43 271 Dörnte B, Kües U, 2012. Reliability in transformation of the basidiomycete Coprinopsis cinerea.
44
45 272 Current Trends in Biotechnology and Pharmacy 6: 340–355.
46 273 Evans HC, Bezerra JL, Barreto RW, 2013. Of mushrooms and chocolate trees: aetiology and
47 274 phylogeny of witches’ broom and frosty pod diseases of cacao. Plant Pathology 62: 728–
48 275 740; doi:10.1111/ppa.12010.
49
50
276 Evans HC, Holmes KA, Phillips W, Wilkinson MJ, 2003a. What’s in a name: Crinipellis, the
51 277 final resting place for the frosty pod rot pathogen of cocoa? Mycologist 16: 148–152;
52 278 doi:10.1017/S0269915X02004093.
53 279 Evans HC, Holmes KA, Reid AP, 2003b. Phylogeny of the frosty pod rot pathogen of cocoa.
54 280 Plant Pathology 52: 476–485; doi:10.1046/j.1365-3059.2003.00867.x.
55
56 281 Evans HC, Stalpers JA, Samson RA, Benny GL, 1978. On the taxonomy of Monilia roreri, an
57 282 important pathogen of Theobroma cacao in South America. Canadian Journal of Botany
58 283 56: 2528–2532.
59
60 9
61
62
63
64
65
 1
2
3
4 284 Fries N, 1978. Basidiospore germination in some mycorrhiza-forming hymenomycetes.
5
6 285 Transactions of the British Mycological Society 70: 319–324; doi:10.1016/S0007-
7 286 1536(78)80128-4.
8 287 Griffith GW, Nicholson J, Nenninger A, Birch RN, Hedger JN, 2003. Witches’ brooms and
9 288 frosty pods : Two major pathogens of cacao. New Zealand Journal of Botany 41: 423–
10
11
289 435.
12 290 Grisales Ortega SP, Kafuri LA, 2007. Análisis de variabilidad genética en Moniliophthora roreri
13 291 con AP-PCR y RAPD en Antioquia, Colombia. Revista Colombiana de Biotecnología IX:
14 292 15–32.
15 293 Ingold CT, 1980. Mycelium, oidia and sporophore initials in Flammulina velutipes. Transactions
16
17 294 of the British Mycological Society 75: 107–116; doi:10.1016/S0007-1536(80)80200-2.
18 295 Kapuscinski J, 1995. DAPI: a DNA-specific fluorescent probe. Biotechnic & Histochemistry 70:
19 296 220–233.
20 297 Kaufert F, 1935. The production of asexual spores by Pleurotus corticatus. Mycologia 27: 333–
21 298 341.
22
23 299 Kemp RFO, 1980. Production of oidia by dikaryons of Flammulina velutipes. Transactions of
24 300 the British Mycological Society 74: 557–560; doi:10.1016/S0007-1536(80)80056-8.
25 301 Kertesz-Chaloupková K, Walser PJ, Granado JD, Aebi M, Kües U, 1998. Blue light overrides
26 302 repression of asexual sporulation by mating type genes in the basidiomycete Coprinus
27
28
303 cinereus. Fungal Genetics and Biology 23: 95–109; doi:10.1006/fgbi.1997.1024.
29 304 Kirschner R, Lee IS, Chen CJ, 2013. Ovularia puerariae Sawada is the hyphomycetous
30 305 anamorph of a new Marasmius species on living leaves of kudzu (Pueraria montana,
31 306 Fabaceae). Mycologia 105: 781–792; doi:10.3852/12-285.
32 307 Kües U, Polak E, Bottoli APF, Hollenstein M, Walser PJ, Boulianne RP, Hermann R, Aebi M,
33
34 308 2002. Vegetative development in Coprinus cinereus. In: Osiewacz HD (ed), Molecular
35 309 Biology of Fungal Development. Marcel Dekker, New York, USA, pp 133–163.
36 310 Leaich LL, Papa KE, 1975. Identification of diploids of Aspergillus flavus by the nuclear
37 311 condition of conidia. Mycologia 67: 674–678.
38
39
312 Meinhardt LW, Lacerda Costa GG, Thomazella DPT, Teixeira PJPL, Carazzolle MF, Schuster
40 313 SC, Carlson JE, Guiltinan MJ, Mieczkowski P, Farmer A, Ramaraj T, Crozier J, Davis
41 314 RE, Shao J, Melnick RL, Pereira GAG, Bailey BA, 2014. Genome and secretome
42 315 analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes
43 316 frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases.
44
45 317 BMC Genomics 15: 164–189; doi:10.1186/1471-2164-15-164.
46 318 Moore D, Robson GD, Trinci APJ, 2011. 21st century guidebook to fungi. Cambridge University
47 319 Press, Cambridge, UK.
48 320 Petersen RH, 1995. There’s more to a mushroom than meets the eye: Mating studies in the
49
321 Agaricales. Mycologia 87: 1–17.
50
51 322 Petersen RH, Hughes KW, Redhead SA, Psurtseva N, Methven AS, 1999. Mating systems in the
52 323 Xerulaceae (Agaricales, Basidiomycotina): Flammulina. Mycoscience 40: 411–426;
53 324 doi:10.1007/BF02464396.
54 325 Phillips DJ, Margosan DA, Mackey BE, 1987. Size, nuclear number, and aggressiveness of
55
56
326 Botrytis cinerea spores produced on media of varied glucose concentrations.
57 327 Phytopathology 77: 1606–1607.
58
59
60 10
61
62
63
64
65
 1
2
3
4 328 Phillips-Mora W, Aime MC, Wilkinson MJ, 2007. Biodiversity and biogeography of the cacao
5
6 329 (Theobroma cacao) pathogen Moniliophthora roreri in tropical America. Plant
7 330 Pathology 56: 911–922; doi:10.1111/j.1365-3059.2007.01646.x.
8 331 Phillips-Mora W, Baqueros F, Melnick RL, Bailey BA, 2015. First report of frosty pod rot
9 332 caused by Moniliophthora roreri on cacao in Bolivia. Plant Pathology 31: 29;
10
11
333 doi:10.1111/j.1365-3059.2006.01378.x.
12 334 Phillips-Mora W, Cawich J, Garnett W, Aime MC, 2006a. First report of frosty pod rot
13 335 (moniliasis disease) caused by Moniliophthora roreri on cacao in Belize. Plant Pathology
14 336 55: 584; doi:10.1111/j.1365-3059.2006.01378.x.
15 337 Phillips-Mora W, Coutiño A, Ortiz CF, López AP, Hernández J, Aime MC, 2006b. First report
16
17 338 of Moniliophthora roreri causing frosty pod rot (moniliasis disease) of cacao in Mexico.
18 339 Plant Pathology 55: 584; doi:10.1111/j.1365-3059.2006.01418.x.
19 340 Phillips-Mora W, Wilkinson MJ, 2007. Frosty pod of cacao: A disease with a limited geographic
20 341 range but unlimited potential for damage. Phytopathology 97: 1644–1647;
21 342 http://dx.doi.org/10.1094/PHYTO-97-12-1644.
22
23 343 Redhead SA, Seifert KA, Vilgalys R, Moncalvo JM, 2000. Rhacophyllus and Zerovaemyces-
24 344 teleomorphs or anamorphs? Taxon 49: 789–798; doi:10.2307/1223980.
25 345 Suárez C, 1971. Estudio del mecanismo de penetración y del proceso de infección de Monilia
26 346 roreri Cif. & Par. en frutos de cacao (Theobroma cacao). BS Thesis, Universidad de
27
28
347 Guayaquil, Ecuador.
29 348 Walther G, Garnica S, Weiß M, 2005. The systematic relevance of conidiogenesis modes in the
30 349 gilled Agaricales. Mycological Research 109: 525–544;
31 350 doi:10.1017/S0953756205002868.
32 351 Walther G, Weiß M, 2008. Anamorphs in the Strophariaceae (Basidiomycota, Agaricales).
33
34 352 Botany 86: 551–566; doi:10.1139/B08-036.
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4 353 Figure legends
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7 354 Fig. 1 – Bright-field (left) and fluorescent (right) microscopy of vegetative and conidiogenous
8 355 hyphae in Moniliophthora roreri isolates. A: DIS 371. B, C: CBS 138632. D: CBS 138631. E:
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10 356 CBS 138635. F: CBS 138636. Notice swelling of conidiogenous cells (Arrows on B and E).
11 357 Bars: 20 μm.
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14 358 Fig. 2 – Bright-field and fluorescent microscopy showing the rhexolytic secession of conidia in
15 359 Moniliophthora roreri isolates. A, B: CBS 138631. C: CBS 138632. D: DIS 371. White arrows
16 360 indicate plasma-free compartments; black arrows indicate binucleate cells. Bars: 20 μm.
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19 361 Fig. 3 – Bright-field and fluorescent microscopy of binucleate and mononucleate conidia from
20 362 Moniliophthora roreri isolates. A: DIS 371. B, C: CBS 138632. D: CBS 138631. E: CBS
21 363 138635. F: CBS 138636 (note trinucleate conidium indicated by black arrows). Bars: 20 μm.
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364 Fig. 4 – Average diameter (AD) and plane area of binucleate (B) and mononucleate (M) conidia
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25 365 from Moniliophthora roreri isolates DIS 371, CBS 1382632, CBS 1382631, and CBS 1382635.
26 366 Values with different letters in the same row differ significantly (Tukey HSD tests, p < 0.001).
27 367 Error bars represent the standard deviations.
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30 368 Fig. 5 – Bright-field and fluorescent microscopy of spore germination of Moniliophthora roreri
31 369 isolate CBS 1382634. Binucleate (A and B) and mononucleate (C and D) spores produce
32 370 monokaryotic hyphae. Bars: 20 μm.
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371 Fig. 6 – Model of thallic rhexolytic conidiogenesis in Moniliophthora roreri. A: Homokaryotic
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36 372 hyphae with varying number of nuclei per cell. B: plasma contraction of hyphal cells and
37 373 formation of plasma-free compartments (black arrows). C: swelling of conidiogenous cells
38 374 during maturation of conidia (grey arrows). D: conidia secession (white arrows), with a potential
39 375 mitotic division during conidia formation. E: mature conidia.
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2 TABLES
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4 380 Table 1 – Origin, host, collection year and variety of Moniliophthora roreri isolates used.
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6 Isolate Origin Host Varietya Single Spore Isolation
7 DIS 371 Esmeraldas, Ecuador T. gileri gileri Yes
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9 CBS 138632 Pichucalco, Mexico T. cacao roreri Yes
10 CBS 138631 Los Ríos, Ecuador T. cacao roreri Yes
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CBS 138635 Cuzco, Peru T. cacao roreri Yes
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13 CBS 138636 Tocache, Peru T. cacao roreri No
14 CBS 138634 Huánuco, Peru T. cacao roreri No
15 a
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381 Variety based on host fide Evans et al. (2003a).
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4 383 Table 2 – Dimensions of the binucleate (B) and mononucleate (M) spores and their frequency in each isolate of Moniliophthora roreri
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6 Lengtha Width Marascuilo Analysis
7 Nuclear
Isolate Variety Proportion
8 condition Avg. Min. Max. Avg. Min. Max. Freq. Total
9 of Bb
10 B 9.8 7.2 13.0 8.2 5.9 10.5 28
11 DIS 371 gileri 59 0.47a
12 M 7.8 5.8 10.7 6.9 4.9 10.3 31
13 B 11.9 5.7 17.0 7.6 4.7 10.2 21
14 CBS 138632 roreri 58 0.36ab
15 M 8.8 6.2 14.1 6.9 4.4 9.4 37
16 B 9.4 6.1 15.4 8.4 6.4 13.5 45
17 CBS 138635 roreri 107 0.42a
18 M 7.8 6.0 15.3 7.0 5.5 9.6 62
19 B 10.2 6.5 18.6 7.9 4.6 10.6 91
20 CBS 138631 roreri 362 0.25b
21 M 7.7 5.4 18.4 6.7 3.7 9.6 271
22 384 a
Avg. =Average; Min. = Minimum observed value; Max. = Maximum observed value
23 385 b
Proportion of binucleate spores that have different letters differ significantly according to the Marascuilo test (p < 0.0001)
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Figure 1
FIGURES
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