Type of the Paper (Article, Review, Communication, etc.

) 1

Growth, pathogenicity and fertility as fitness characters in the 2

tree pathogen Ceratocystis albifundus 3

Vinolia N. Danki1, Nokuthula P. Mchunu2, Magriet A. van der Nest1, Lieschen De Vos1, Benedicta S. Swalarsk- 4
Parry1, Felix Fru1, P. Markus Wilken1, Mike J. Wingfield1*, Brenda D. Wingfield1 and Emma T. Steenkamp1 5

1 Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute 6
(FABI), University of Pretoria, Pretoria, 0002, South Africa. 7
2 National Research Foundation, Pretoria, South Africa. 8
* Correspondence: magriet.vandernest@fabi.up.ac.za 9

Abstract: Ceratocystis albifundus is an African fungus that does not usually cause disease on the range 10
of woody hosts in its native range. However, on introduced Acacia species and orchard-grown Protea 11
cynaroides, it represents an economically significant pathogen. Despite the ecological success of this 12
fungus, few studies have explored the components of its biological fitness. For example, it has been 13
suggested that sexual fertility is an important aspect of the fitness of C. albifundus, because self-fertile 14
isolates generally had higher growth rates and aggressiveness than their self-sterile counterparts. 15
However, all these previous studies were based on small numbers of isolates, thereby precluding 16
reasonable inferences regarding wider populations. The aim of this study was therefore to explore 17
how two commonly used measures of fitness (growth in culture and pathogenicity) vary across a 18
wide collection of isolates. We also investigated whether these are potentially linked to the 19
geographic origin, fertility and original hosts of the isolates. For this purpose, a diverse collection of 20
58 isolates were subjected to growth studies, pathogenicity tests on Acacia mearnsii, and genome 21
sequencing for scoring their fertility status. Although the isolates varied greatly for all these traits, 22
host of origin and geographic origin were strong predicters of aggressiveness on A. mearnsii. 23
Statistical analyses further separated the isolates into two broad groups based on the disease status 24
of the original tree hosts (i.e., isolates from diseased Acacia and P. cynaroides grouped separately 25
from isolates originating from apparently asymptomatic hosts). These patterns were independent 26
of the fertility status of the isolates, as similar results were obtained when self-sterile isolates were 27
excluded from the analyses. Taken together, the results suggest that growth, pathogenicity and 28
fertility each represent different fitness components, with each being underpinned by distinct 29
mechanisms. These findings thus provide a valuable basis from which to explore the molecular 30
processes shaping the fitness landscape of this important African fungus. 31
Citation: To be added by editorial
staff during production.
Keywords: biological fitness; mycelial growth; pathogenicity; fertility; population-wide variation. 32
Academic Editor: Firstname 33
Lastname

Received: date
Revised: date 1. Introduction 34
Accepted: date Ceratocystis albifundus (Ascomycota) is an African fungus that resides in the 35
Published: date Ceratocystidaceae together with numerous economically important plant pathogens [1,2]. 36
In southern and eastern Africa, C. albifundus infects many different woody plants (e.g., 37
Protea gigantea, Burkea africana and Combretum mole) native to the region without causing 38
Copyright: © 2023 by the authors. any obvious signs of disease [3]. However, on the non-native species Acacia mearnsii and 39
Submitted for possible open access Acacia melanoxylon, and commercially cultivated Protea cynaroides, the fungus is an 40
publication under the terms and aggressive pathogen [3,4]. On these plants it causes canker and wilt disease resulting in 41
conditions of the Creative Commons significant losses to the forestry and cut-flower industries [4,5,6]. 42
Attribution (CC BY) license
Like other members of the Ceratocystidaceae, C. albifundus is closely associated with 43
(https://creativecommons.org/license
insects [1]. In these associations, the fungus usually serves as a source of nutrition for the 44
s/by/4.0/).

Pathogens 2023, 12, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/pathogens

Pathogens 2023, 12, x FOR PEER REVIEW 2 of 12

insects, which in turn assists with dispersal of the fungus. Accordingly, fungi in this 45
family are adapted for insect dispersal such as by producing ascospores in sticky drops 46
borne at the apices of long-necked ascocarps [1]. In the case of C. albifundus, specifically, 47
sap-feeding nitidulid beetles (Coleoptera: Nitidulidae) may also vector the fungus when the 48
insects visit fresh wounds on trees, thus providing it with a means of dispersal [7]. 49
Since its initial discovery in South Africa [8], a substantial body of information on the 50
biology and evolution of C. albifundus has accumulated (e.g., [9,10,11]). In a series of recent 51
studies, Lee et al. [4,12,13] demonstrated that the reproductive biology of C. albifundus 52
represents an essential component of its overall fitness. In filamentous fungi, fitness 53
typically refers to survival and reproductive success in a particular environment [14]. 54
With the aid of mycelial growth assays and pathogenicity tests, Lee et al. [12] showed that 55
compared to self-sterile isolates, self-fertile isolates grow more rapidly and are more 56
aggressive (i.e., aggressiveness is the quantitative component of pathogenicity [15]). They 57
also showed that self-fertile isolates are more prevalent in natural populations and in the 58
progeny produced from sexual interactions [12,13]. Because self-fertile isolates can 59
complete the sexual cycle without compatible mating partners, as is the case for self-sterile 60
isolates [16,17], self-fertile individuals are superior in their ability to produce infective 61
propagules and to rapidly spread or cause epidemics at host population scales [15,18]. 62
This is because self-fertility facilitates the production of ascocarps carrying sticky spore 63
drops to mediate insect-vectored transmission, which in turn affords self-fertile isolates a 64
competitive advantage, ultimately ensuring survival and efficient reproduction of C. 65
albifundus. 66
In general, fungal fitness is dependent on various characters and/or combinations of 67
characters that determine optimal life-history strategies [18]. In addition to reproductive 68
biology, other notable components of fitness may include mycelial growth rate, 69
sporulation, pathogenicity, substrate/host range, symbiotic relationships, and many other 70
factors [19,20,21]. Because these traits are not typically correlated and vary substantially 71
across populations [22], empirical data are needed in order to understand how individual 72
fitness components are linked to the overall growth and survival of species in particular 73
environments [14,23]. Such studies have revealed that some characters may be used as 74
proxies for predicting the fitness and the long-term survival of certain fungi [18,23,24]. In 75
the case of C. albifundus, little is known regarding the variation that might be expected 76
when growth and pathogenicity of isolates from different locations and hosts are 77
compared, because previous studies addressing these questions were based on small 78
numbers of isolates [4,6,12,25]. This was also true for those studies reporting the influence 79
of self-fertility on growth and pathogenicity of C. albifundus [4,12]. 80
The aim of this study was to provide a population-wide framework for C. albifundus 81
in South Africa regarding the variation that could be expected in growth, pathogenicity, 82
and fertility status of the fungus. This was achieved by assembling a large collection of 83
isolates from diverse hosts and locations, which were then used to investigate fertility 84
status, growth in culture, and pathogenicity on A. mearnsii. We also investigated whether 85
the growth and pathogenicity data could reflect possible structuring of populations 86
according to the geographic origin and source/host from which isolates were obtained and 87
their fertility status. Finally, to identify possible proxies for the overall fitness C. albifundus, 88
we investigated whether any of the traits examined are correlated with one another. 89

2. Materials and Methods 90

2.1. Fungal isolates and routine culturing 91

A collection of 58 C. albifundus isolates were included in this study. These were 92

isolated from a wide range of hosts including both native, and non-native plants, as well 93
as two isolates obtained from beetle species (Table S1). The isolates originated from a wide 94
geographic range in South Africa, including various locations in the summer rainfall (i.e., 95
in KwaZulu-Natal, Gauteng, Limpopo, and Mpumalanga provinces) and winter rainfall 96
Pathogens 2023, 12, x FOR PEER REVIEW 3 of 12

(Western Cape Province) regions of South Africa. These isolates were routinely cultured 97
in the dark at 25°C for 7-14 days on 90 mm Petri dishes containing malt extract agar (MEA) 98
medium. This contained 2% (w/v) malt extract (Biolab, Midrand, South Africa) and 2% 99
(w/v) bacteriological agar (Biolab). All isolates are maintained in the culture collection 100
(CMW) of the Forestry and Agricultural Biotechnology Institute (FABI) of the University 101
of Pretoria (South Africa). 102

2.2. Fertility status assay 103

The fertility status of the isolates was assayed using their genome sequences. To 104
achieve this, high quality DNA was extracted from 2-week-old cultures using the 105
cetyltrimethylammonium bromide (CTAB) method [26]. The DNA was then used to 106
obtain single reads of 400 bp using the Ion-Torrent™ Ion S5™ system and Ion 530 Chip 107
Kit at the Central Analytical Facility (University of Stellenbosch, Stellenbosch, South 108
Africa). The reads were filtered for quality with Trimmomatic v.0.39 using default settings 109
[9,27] and assembled using the published sequence for isolate CMW4068 as reference [11]. 110
This was done with the “bwa mem” command in BWA (Burrows-Wheeler Aligner) 111
v.0.7.17 by setting the “ploidy” function to haploid and using default settings for the 112
remaining parameters [28,29]. The BWA output was then used by SAMtools v.1.9 to create 113
fasta files for individual genome assemblies [29], which we explored for the presence of 114
the MAT1-2-1 gene. This is typically encoded by self-fertile isolates and not self-sterile 115
isolates [16]. The known MAT1-2-1 sequence was obtained from GenBank® [30] using the 116
accession number KF033902 [16]. It was then searched against the translated sequences of 117
each genome with the aid of local tBLASTn searches in CLC Genomics Workbench v.10 118
(CLC bio, Aarhus, Denmark). 119

2.3. Mycelial growth in culture 120

For each C. albifundus isolate, the radial mycelial growth on MEA was determined as 121
previously described [12]. For each isolate, a sterile 5 mm cork borer was used to take six 122
mycelial plugs from the actively growing margin of a 2-week-old MEA culture, and then 123
transferring it to the centers of six separate sterile 90 mm Petri dishes containing MEA. 124
The plates were sealed with Parafilm M® (Merck; Darmstadt, Germany), after which three 125
were incubated at 25°C and an equal number were at incubated 30°C all in the dark. 126
Following 21 days of incubation, colony diameter was recorded by taking the means of 127
two perpendicular measurements (mm) for each plate. The entire experiment was 128
performed twice. 129

2.4. Pathogenicity tests 130

The relative aggressiveness of isolates was assessed in a greenhouse inoculation 131
study using 2-year-old A. mearnsii trees as described previously [31]. For each isolate, a 132
sterile 7 mm cork borer was used to take a mycelial plug from the actively growing margin 133
of a 2-week-old MEA culture and placing it with the mycelium side facing towards a 134
wound made on the plant stems. The wounds were made on the stems 20 cm above 135
ground level using a sterile 10 mm cork borer to remove the bark and expose the 136
underlying cambial tissue. Inoculation wounds were sealed with Parafilm M® to keep the 137
plugs in position and to prevent desiccation and cross contamination. For the control 138
treatment, inoculations were performed in the same way but using sterile MEA plugs 139
rather than fungal material. A total of 10 plants were inoculated for each isolate and equal 140
number for the controls. After being maintained for 3 weeks at 25°C in a greenhouse, the 141
bark around the point of inoculation was removed and the lengths (mm) of the lesions 142
were recorded. The entire experiment was performed twice. 143

2.5. Statistical analyses 144

Pathogens 2023, 12, x FOR PEER REVIEW 4 of 12

Data for mycelial growth in culture and aggressiveness were subjected to various 145
analyses. The mean colony diameter and mean lesion length measurements for each 146
isolate were visualized using histograms. The growth and pathogenicity data were also 147
subjected to various statistical analyses to determine if these traits were significantly 148
linked to the disease status (symptoms or no symptoms) of the host and/or fertility status 149
(self-fertile or sterile) of the isolates. For the analyses involving geographic origin, the 150
limited number of isolates originating from different provinces in the summer rainfall 151
region of South Africa precluded reliable statistical treatments. To account for this, isolates 152
were grouped as being from the winter rainfall region (i.e., Western Cape Province) or 153
from the summer rainfall region (i.e., Kwa-Zulu Natal, Mpumalanga, Limpopo and 154
Gauteng). Additionally, to exclude the possible impact that isolate fertility status might 155
have on the patterns observed, all analyses were repeated on datasets from which isolates 156
scored as self-sterile were removed. 157
To test for normality in the growth and pathogenicity datasets, the Kolmogorov- 158
Smirnov test was used. For comparison of means, the data were subjected to one-way 159
ANOVA (analysis of variance) or two-tailed Student’s t-test using a confidence level of 160
95%. Here the null hypothesis assumed no differences between the means for the different 161
groups. Tukey’s Honestly Significant Difference (HSD) tests were performed to identify 162
groups that differed significantly from each other. All ANOVAs and Tukey’s HSD 163
analyses were performed using the online tools available respectively at 164
https://acetabulum.dk/anova.html. The Kolmogorov-Smirnov and Student’s tests were 165
performed with the tools available at https://www.socscistatistics.com. 166
A Chi squared (X2) test was used to determine whether the observed ratio of self- 167
fertility to sterility departed significantly (α = 0.05) from the data presented by Lee et al. 168
[13] using the online tool available at https://www.socscistatistics.com. The growth and 169
pathogenicity data were also subjected to Pearson’s product-moment correlation analyses. 170
Correlations between these continuous data and the categorical data were made 171
determining the point-biserial correlation coefficient (rpbc). The latter test is a derivation of 172
Pearson’s product-moment correlation analysis where one of the variables is continuous 173
and the other dichotomous. Categorical data included fertility status (self-fertile or self- 174
sterile), and original host’s disease status (symptoms or no symptoms). To explore a 175
possible correlation between different sets of categorical data, we used the Phi (Φ) 176
coefficient, which is analogous to Pearson’s product-moment correlation coefficient 177
(rPearson) but that estimates the degree of association between two binary variables. 178
Additionally, correlations among the fertility status of isolates, as well as the identity of 179
the host, disease status and geographic origin were explored using Principal Component 180
Analysis (PCA) with SRplot (https://www.bioinformatics.com.cn/en). The latter software 181
was also used to generate biplots in order to graphically display Principal Component 182
(PC) scores and how variable vectors are projected onto the PCs. 183

3. Results 184

3.1. Fertility status assay 185

The genomes of 45 isolates examined encoded the MAT1-2-1 gene and were scored 186
as self-fertile (Figure 1). The gene was absent from 13 genomes, and those isolates were 187
scored as self-sterile. The self-sterile isolates originated from diverse hosts, i.e., P. 188
cynaroides (n=7), as well as Acacia species (n=2) and different native tree species (n=4) from 189
various locations in the summer rainfall area of South Africa (Table S1). Nevertheless, the 190
observed ratio of ca. 4:1 self-fertile to self-sterile isolates of C. albifundus differed 191
significantly (X2 = 8.6568; P < 0.05) from the 1:1 ratio that would be expected following 192
Mendelian segregation. However, it did not differ significantly (X 2 = 2.1913; P = 0.1388) 193
from the average ratio (ca. 7:1) reported previously for instances where self-fertility 194
dominated among isolates collected from native hosts or P. cynaroides orchards [13]. 195
Pathogens 2023, 12, x FOR PEER REVIEW 5 of 12

Isolate Host Origin Fer lity Pathogenicity Growth: 25°C and 30°C
CMW22302 Nitidulid beetle
CMW21473 Nitidulid beetle
CMW42100 Nitidulid beetle
CMW42102 Staphilinid beetle
CMW23839 Terminalia sericea
CMW17620 Terminalia sericea
CMW37313 Combretum zeyheri
CMW21146 Vachellia grandicornuta
CMW41550 Lannea schweinfurthii
CMW21150 Senegalia nigrescens
CMW37312 Terminalia sericea
CMW17628 Faurea saligna
CMW40625 Protea gaguedi
CMW41580 Terminalia sericea
CMW41552 Lannea schweinfurthii
CMW42119 Terminalia sericea
CMW41551 Lannea schweinfurthii
CMW42123 Terminalia sericea
CMW42120 Terminalia sericea
CMW42118 Terminalia sericea
CMW41601 Terminalia sericea
CMW37949 Combretum apiculatum
CMW23823 Acacia mearnsii
CMW4075 Acacia mearnsii
CMW4084 Acacia mearnsii
CMW42103 Acacia melanoxylon
CMW4090 Acacia mearnsii
CMW42104 Acacia melanoxylon
CMW42117 Acacia melanoxylon
CMW42450 Protea cynaroides
CMW42415 Protea cynaroides
CMW42478 Protea cynaroides
CMW42444 Protea cynaroides
CMW42446 Protea cynaroides
CMW42409 Protea cynaroides
CMW42428 Protea cynaroides
CMW43682 Protea cynaroides
CMW42474 Protea cynaroides
CMW42417 Protea cynaroides
CMW42420 Protea cynaroides
CMW42445 Protea cynaroides
CMW42447 Protea cynaroides
CMW42442 Protea cynaroides
CMW42476 Protea cynaroides
CMW42435 Protea cynaroides
CMW42408 Protea cynaroides
CMW42429 Protea cynaroides
CMW42423 Protea cynaroides
CMW42436 Protea cynaroides
CMW42419 Protea cynaroides
CMW42425 Protea cynaroides
CMW43681 Protea cynaroides
CMW42477 Protea cynaroides
CMW42432 Protea cynaroides
CMW42437 Protea cynaroides
CMW42416 Protea cynaroides
CMW43683 Protea cynaroides
CMW42438 Protea cynaroides
Native Winter rainfall Self-fertile
0
0 50
50 100
100 150
150 00 20
20 40
40 60
60
Planted Summer rainfall Self-sterile Mean lesion length (mm) Mean colony diameter (mm)
196
Figure 1. Information on the host, origin, fertility status of the Ceratocystis albifundus isolates 197
examined, as well as the mean length of lesions they induced on 2-year-old Acacia mearnsii trees and 198
the mean diameter of colonies after 7 days of growth at 25°C and 30°C on malt extract agar medium. 199
In the pathogenicity pane, isolates that did not differ significantly from the “no fungus” control 200
treatment are indicated in grey. 201
Pathogens 2023, 12, x FOR PEER REVIEW 6 of 12

3.2. Mycelial growth in culture 202

The mean colony diameter following two weeks of growth at 25°C and 30°C varied 203
widely across the 58 C. albifundus isolates examined (Figure 1). These ranged from 14 mm 204
to 54 mm at 25°C and 21 mm to 55 mm at 30°C. Most isolates grew faster at the higher 205
temperature, but there were some exceptions. Similar results were observed from the 206
repeat experiment. Both datasets were normally distributed with Kolmogorov-Smirnov D 207
statistic values of 0.08581 (P = 0.74529) for the 25°C growth data and 0.10411 (P = 0.51145) 208
for the 30°C data. Accordingly, ANOVA and Student’s t-tests were used to evaluate 209
whether the means for specific groups of isolates differed significantly (Table S2 and S3). 210

211
Figure 2. Histograms showing mean mycelial growth at 25°C (yellow) and 30°C (green), as well as 212
and mean lesion length (blue) for all 58 of the C. albifundus isolates examined in this study (a), and 213
for the 45 self-fertile isolates only (b). The isolates are grouped based on their host species, 214
geographic origin, whether they were isolated from healthy or diseased hosts. Bars represent 215
standard deviation, while the different letters within each set of results indicate significant (P <0.05) 216
differences between or among means (see Tables S2 and S3 for details of statistical analyses). 217

Isolates were grouped in terms of their fertility status, host of origin and geographic 218
origin, as well as their ability to cause disease on A. mearnsii and the disease status of the 219
host of origin. However, no significant differences were observed between or among the 220
Pathogens 2023, 12, x FOR PEER REVIEW 7 of 12

colony diameter means for any of the examined groups of isolates at either temperature 221
(Figure 2A). For the 46 self-fertile isolates the growth data were also normally distributed 222
at both 25°C (D = 0.1173; P = 0.7430) and 30°C (D = 0.1236; P = 0.4350). As with the entire 223
collection of isolates, no significant differences were detected among or between any of 224
the evaluated groups of means (Figure 2B; Table S2 and S3). The only exception was for 225
the comparison between geographic origin and the host of origin. Self-fertile isolates from 226
Acacia species as well as the native hosts grew significantly (P < 0.05) more rapidly at 25°C 227
than those from P. cynaroides (Table S1). This was also reflected by the winter rainfall 228
isolates (mainly obtained from P. cynaroides and A. melanoxylon) growing significantly (P 229
< 0.05) more slowly than those from the summer rainfall region (mainly isolates obtained 230
from native hosts and from A. mearnsii) (Table S3). 231

3.3. Pathogenicity tests 232

The mean lesion lengths obtained for the 58 C. albifundus isolates inoculated onto A. 233
mearnsii saplings ranged from 16 mm for isolate CMW42450 to 134 mm for isolate 234
CMW4090 (Figure 1), with similar overall patterns observed from the repeat experiment. 235
The data obtained from the pathogenicity assay were normally distributed (D = 0.0740, P 236
= 0.8851), and the ANOVA results suggested significant (P < 0.05) differences between the 237
recorded mean lesion lengths (Table S2). The latter also showed that 13 of the isolates 238
produced lesions that were not significantly different from the control treatment that was 239
characterized by a mean lesion length of 12 mm (see isolates indicated in grey in the 240
“pathogenicity” pane of Figure 1). 241
ANOVA and Student’s t-tests revealed significant (P < 0.05) differences among or 242
between groups of means when isolates were organized based on their host of origin, as 243
well as whether they were from disease symptoms or not (Tables S2 and S3). Isolates 244
obtained from the tissues of symptomatic Protea and Acacia species produced significantly 245
longer lesions than isolates originating from native hosts lacking signs of disease (Figure 246
2). The same was also true when isolates were grouped based on geographic origin, where 247
isolates from the winter rainfall region mostly formed significantly (P < 0.05) longer 248
lesions than those from the summer rainfall region. Additionally, the self-fertile isolates 249
produced significantly longer lesions than the self-sterile isolates (Figure 3; Table S3). 250
The dataset containing mean lesion lengths for the 45 self-fertile isolates were 251
normally distributed (D = 0.0695; P = 0.9650), with ANOVA and Student’s t-tests showed 252
significant (P < 0.05) differences among and between various groups of means examined 253
(Tables A2 and A3). Overall, similar patterns were observed as those obtained with the 254
entire isolate collection. Self-fertile isolates originating from Acacia gave rise to 255
significantly (P < 0.05) longer lesions than those from the native hosts (Figure 2). Self- 256
fertile isolates obtained from diseased plant tissue and from the summer rainfall region 257
also produced significantly longer lesions that those obtained from asymptomatic plants 258
and from the winter rainfall region (Figure 2). 259

3.4. Correlation analyses 260

A moderate positive correlation (rPearson = 0.5458, P < 0.00001) was observed between 261
mycelial growth at 25°C and 30°C for the 58 C. albifundus isolates (Table S4). However, 262
there was no significant correlation between the pathogenicity data and mycelial growth 263
at either 25°C (rPearson = -0.0725, P = 0.5769) or 30°C (rPearson = 0.071, P = 0.5741). No significant 264
correlation was detected when any of these continuous variables were compared with the 265
binary traits such as fertility (self-fertile or self-sterile), and whether the isolates were 266
associated with disease or from wounds in the absence of disease. Additionally, there was 267
no significant correlation among any of the binary variables. 268
The results for the various correlation analyses with only the data for the self-fertile 269
isolates were mostly similar to those obtained using all isolates (Table S4). Again, a 270
moderate positive correlation (rPearson = 0.4546, P = 0.0017) was observed between the 271
Pathogens 2023, 12, x FOR PEER REVIEW 8 of 12

growth of self-fertile isolates at the two temperatures, and neither of the latter data sets 272
were correlated with the lesion length data for the self-fertile isolates. Also, as with the 273
entire dataset for both self-sterile and self-fertile strains, growth at 25°C was weakly 274
correlated with pathogenicity to A. mearnsii (Table S4). 275
To explore possible correlations between the pathogenicity, growth, and fertility 276
status of isolates, whether they were associated with disease and considering their 277
geographic origin, we used a PCA biplot to visualize the data for the first two PCs (Figure 278
3). When both self-fertile and self-sterile isolates were included, the two major PCs 279
accounted for 63.6% of the total variability in the data. PCA of this dataset revealed two 280
groups, which contained most isolates from, respectively, native woody hosts lacking any 281
obvious signs of disease, and from plants cultivated commercially (i.e., Acacia species and 282
P. cynaroides) and where they caused disease. This clustering pattern was most strongly 283
affected by the pathogenicity of isolates on A. mearnsii, as well as the disease status and 284
geographic origin of the host (see vectors “Lesion”, “Region” and “Host” in Figure 3A). 285
However, the direction of the “Host” vector was opposite to those of the “Lesion” and 286
“Region” vectors. Mycelial growth and fertility status of the isolates impacted the 287
observed clustering patterns to a lesser extent. A similar pattern was obtained when 288
fertility was excluded as a variable and the PCA conducted on the self-fertile isolates only 289
(Figure 3B). 290

●Asymptomatic host ●Asymptomatic host

A ●Diseased host B ●Diseased host
2.5 2.5
PC2 (explains 28.3% of the total variability)

PC2 (explains 26.4% of the total variability)

0 0

-2.5 -2.5

-2.5. 0 2.5 -2.5. 0 2.5

PC1 (explains 35.3% of the total variability) PC1 (explains 48.7% of the total variability)
291
Figure 3. Biplots of principal component (PC) analyses performed on the pathogenicity, growth, 292
geographic origin, and host data for the C. albifundus isolates included in this study, by either 293
including (a) or excluding (b) fertility status as a variable. Individual points represent PC scores for 294
the observations relative to the first two PCs. The arrows indicate loadings associated with the 295
isolates’ geographic origin (“Region”), hos of origin (“Host”), as well as the lesion length obtained 296
in the pathogenicity test (“Lesion”), mycelial growth at 25°C and 30°C (“Growth25” and 297
“Growth30”). Prediction ellipses are such that with probability 0.95, a new observation from the 298
same group will fall inside the ellipse. 299

4. Discussion 300

This study provides the first comprehensive view of how pathogenicity and growth 301
of C. albifundus might be expected to vary across populations. All previous studies (e.g., 302
[3,12,32]) exploring these traits utilized only a small number of C. albifundus isolates. Using 303
a collection of 58 isolates originating from different hosts, those associated with disease or 304
only occurring on wounds in the absence of disease, and isolates from different regions of 305
South Africa, we showed that C. albifundus varied widely in its aggressiveness on A. 306
mearnsii and its mycelial growth rate in culture at 25°C and 30°C. Surprisingly, none of 307
these patterns were linked to whether the isolates where self-fertile or self-sterile, as the 308
Pathogens 2023, 12, x FOR PEER REVIEW 9 of 12

exclusion of self-sterile isolates yielded similar results. Exploration of these patterns might 309
thus hold valuable clues as to how these traits contribute to the overall fitness of C. 310
albifundus [14,23]. 311
The phenotypic separation of isolates from the winter and summer regions is 312
consistent with what has been reported from recent microsatellite-based population 313
genetic studies [4]. Here, isolates from the winter rainfall region grew significantly more 314
slowly at 25°C, while they induced significantly longer legions on A. mearnsii than those 315
from the summer rainfall region. In their study, Lee et al. [4] argued that the occurrence 316
of C. albifundus in the winter rainfall region is likely due to recent introduction(s) into that 317
area, combined with host range expansions and/or host shifts [4,33,34]. Such a scenario is 318
in agreement with the notion that fungi from “new encounter” hosts (in this case P. 319
cynaroides and A. melanoxylon) are often more virulent or aggressive than in their native 320
range (e.g., [35,36]). Therefore, the introduction(s) giving rise to the population in the 321
winter rainfall region likely included C. albifundus genotypes capable of surviving there 322
and overcoming the defense mechanisms of some plant species occurring there [15,35]. 323
Overall, isolate aggressiveness on A. mearnsii was strongly correlated with the 324
geographic origin of C. albifundus and the hosts from which isolates originated. Isolates 325
from the summer rainfall region or from native hosts induced significantly shorter lesions 326
on 2-year-old A. mearnsii trees than those from the winter rainfall region, as well as those 327
isolated from A. melanoxylon, A. mearnsii and orchard-grown P. cynaroides. In part, this was 328
not unexpected because geographic differentiation in microbial populations is often 329
linked to the climatic conditions in particular regions, which influence not only the 330
distribution of the microbe but also that of its plant host [37,38]. However, the fact that C. 331
albifundus lacks aggressiveness or is less aggressive towards the woody hosts with which 332
it shares a native range [3,4,12,33,34,39], probably also reflects its co-evolutionary 333
relationship with these plants [40,41]. By contrast, its aggressiveness to Acacia and 334
orchard-grown P. cynaroides is a consequence of these plants not possessing suitable 335
mechanisms for fending it off [4,33,34]. Therefore, the fitness of both C. albifundus and its 336
various plant hosts are likely governed by their co-evolutionary relationships, as well as 337
their geographic occurrence and the climatic conditions associated with it. 338
The ratio of self-fertile to self-sterile isolates in the collection of C. albifundus isolates 339
utilized in this study was strongly skewed towards the self-fertility, which is in agreement 340
with previous reports [12,13]. This situation might arise when fewer ascospores bearing 341
the self-sterile trait originate in an ascus and/or when ascospores bearing the trait fail to 342
germinate [12]. Our findings were also consistent with previous suggestions that self- 343
sterile isolates are less aggressive than their self-fertile counterparts [12,13]. However, 344
with a few exceptions, the self-fertile isolates mostly did not grow more slowly than the 345
self-sterile isolates (see Supplementary Figure S1). Nevertheless, why self-sterility is 346
retained in C. albifundus populations remains unclear, but the presence of self-sterile 347
isolates potentially promotes outcrossing, thereby allowing the fungus to fully exploit the 348
benefits of sexual recombination [42,43,44]. The presence of self-sterile isolates in 349
populations could thus mediate re-assortment of advantageous alleles, or lead to the 350
introduction of new alleles, while self-fertile isolates ensure through selfing the 351
multiplication and transmission of advantageous allelic combinations that are locally 352
adapted without the process of outcrossing [20,45,46,47]. 353
The results of this study suggest that fertility, growth, and pathogenicity each 354
represent distinct components of fitness that contribute separately towards optimizing the 355
life-history strategy of C. albifundus. The distribution patterns associated with these traits 356
were all different from one another and none of them were correlated, as has been found 357
in other fungi [18,48]. Given the complexities associated with the life cycle of C. albifundus 358
(i.e., mitotic, and meiotic reproduction involving both selfing and outcrossing) [14,49], it 359
also might be that different types of interactions among these and other traits determine 360
the overall fitness of the organism (e.g., [19,23]). For example, in the case of saprophytic 361
filamentous fungi, rapid mycelial growth and high levels of spore production are often 362
Pathogens 2023, 12, x FOR PEER REVIEW 10 of 12

inversely correlated, thus representing fitness trade-offs for ensuring either exploration of 363
nearby nutritional resources or dispersal to new sites [14,19,50]. The individual 364
contributions of growth, fertility, and pathogenicity to the fitness of C. albifundus therefore 365
requires further investigation. It should also be examined how these traits might be 366
influenced by environmental factors or by other organisms the fungus might interact with. 367
This is particularly true given the impact of plant host and geography on the patterns 368
observed here, as well as the potential involvement of insects and other fungi in the 369
general ecology of C. albifundus (e.g., [7,25]). 370
Taken together, our findings provide a useful resource to decipher the molecular 371
basis and mechanisms underlying the traits examined. For example, the data for both 372
growth in culture and aggressiveness on A. mearnsii displayed continuous distributions, 373
irrespective of the isolates’ fertility status, their host of origin and geographic origin. This 374
suggests that both traits are quantitative and governed by multiple loci and/or genes. 375
Similar patterns have been reported in other fungi for growth in culture (e.g., [11,51,52]) 376
and pathogenicity (e.g., [53,54]). The general lack of correlation between the traits further 377
suggest that different sets of loci likely determine the variation observed (e.g., [51,55]). 378
These data, combined with the genome sequences generated in the current study, would 379
thus allow for comprehensive analyses (e.g., comparative genomics and Genome Wide 380
Association Studies) of the molecular nature of the traits and subsequent inference of the 381
contribution of each trait to the overall fitness of C. albifundus. This could furthermore 382
reveal avenues to manage pathways of movement of the pathogen and for the diseases it 383
causes. 384

Supplementary Materials: The following supporting information can be downloaded at: 385
www.mdpi.com/xxx/s1, Table S1: Geographic origin and host of C. albifundus isolates screened for 386
growth rate and pathogenicity; Table S2: ANOVA results for the lesion length data and mycelial 387
growth at 25°C and 30°C when isolates are grouped according to their original plant host species; 388
Table S3: Results of Student’s t-tests for comparisons performed in this study; Table S4: Results of 389
correlation analyses between the respective continuous and categorical variables examined in this 390
study. 391

Author Contributions: Conceptualization: V.D., M.A.vdN., E.T.S.; Methodology: V.D., N.P.M., 392
E.T.S.; Formal analysis: V.D., B.S.S., F.F., E.T.S.; Resources: M.A.vdN., B.D.W., M.J.W.; Data curation: 393
L.dV., F.F., N.P.M, P.M.W.; Writing—original draft preparation: V.D., N.P.M., F.F., L.dV., 394
M.A.vdN., E.T.S.; Writing—review and editing: B.D.W., P.M.W., M.J.W., E.T.S.; Supervision: N.P.M, 395
L.dV, B.D.W., M.A.vdN., E.T.S.; Project administration: M.A.vdN; Funding acquisition: M.A.vdN. 396
All authors have read and agreed to the published version of the manuscript. 397

Funding: We thank the Department of Science and Innovation (DSI) - National Research Foundation 398
(NRF) Centre of Excellence in Tree Health Biotechnology and SARChI Chair in Fungal Genomics 399
for financial support. 400

Data Availability Statement: The data presented in this study are available on request from the 401
corresponding author. 402

Conflicts of Interest: The authors declare no conflict of interest. 403

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