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A. F. Solarte et al. Plant Disease 1
Diversity of Neopestalotiopsis and Pestalotiopsis spp., Causal Agents of Guava Scab in 1
Colombia 2
3
Fernando Solarte and Carlos German Muñoz, Universidad Nacional de Colombia–Palmira, 4
Colombia, Sajeewa S. N. Maharachchikumbura, Department of Crop Sciences, College of 5
Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khod 123, Oman, 6
and Elizabeth Álvarez, International Center for Tropical Agriculture (CIAT), Palmira, Colombia. 7
8
Corresponding author: Elizabeth Álvarez. 9
E-mail: e.alvarez@cgiar.org 10
11
12
GenBank accession numbers: KR493520 to KR493741 13
14
All authors have reviewed the manuscript and approve its submission to Plant Disease. The 15
manuscript is not being submitted elsewhere. 16
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A. F. Solarte et al. Plant Disease 2
Abstract 20
Common guava (Psidium guajava L.) is a fruit tree of global economic importance. It is grown in 21
Asia, South and Central America, and Hawaii for its exquisite aroma and flavor, and nutritional 22
and medical properties. However, guava production is limited by guava scab, caused by fungi in 23
the Pestalotiopsis genus. Characteristic symptoms of guava scab are corky, ovoid or round lesions 24
on fruit surfaces. These lesions may thicken, affecting the flesh below, and reducing fruit quality 25
and commercial value. We characterized 81 isolates isolated from guava scab lesions on guava 26
leaves and fruits in different regions of Colombia, and identified them as Pestalotiopsis and 27
Neopestalotiopsis spp. We analyzed the morphology, pathogenicity, and genetic diversity of the 28
isolates based on the sequences of the internal transcribed spacer (ITS), β-tubulin, and the 29
elongation factor genes. Isolates were morphologically, pathogenically and genetically diverse, but 30
the diversity did not correlate with geographical origin or guava cultivar or tissue from which the 31
isolates were recovered. Selected monosporic isolates included in the multiple-gene analysis, were 32
identified as belonging to two genera: Neopestalotiopsis (65 isolates with versicolorous conidia) 33
and Pestalotiopsis (4 isolates with concolorous conidia). 34
35
36
37
38
39
40
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42
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A. F. Solarte et al. Plant Disease 3
Introduction 44
Guava (Psidium guajava L.) is a small tree that belongs to the genus Psidium (Pérez et al. 45
2008). The center of origin for guava extends from Mexico to Peru but it is cultivated in most 46
tropical and subtropical countries (Pérez et al. 2008). Guava grows in soils of differing textures, 47
drainage, and pH (between 4.5 and 9.4). It can grow in regions with high mean annual rainfall or in 48
areas subject to drought (Keith et al. 2006). 49
In Colombia, guava is usually consumed fresh or processed into a firm, sweet pulp known 50
as bocadillo that is used for juices and jams (Melgarejo et al. 2010). The fruit is a good source of 51
calcium, iron, and phosphorus (Pachanawan et al. 2008). It also provides vitamin A, ascorbic acid 52
(vitamin C), three essential amino acids (tryptophan, lysine, and methionine), and three vitamin B 53
compounds (thiamine or B1, riboflavin or B2, and niacin or B3) (Pérez et al. 2008). In Colombia, 54
guava production averaged 1.3 million tons per year over the past five years (Romero et al.2014). 55
Guava production is socioeconomically important in Colombia, providing more than 4,800 56
jobs in places such as La Hoya del Río Suárez, Colombia (Rodríguez-Borray and Rangel-Moreno 57
2005). Guava is an important commodity for domestic markets, but it also has great export 58
potential. However, guava production is subject to various production constraints including pests 59
and diseases. Guava scab, also known as pestalotiopsis or nailhead rust, is the most important 60
disease constraint to guava cultivation in Colombia, affecting fruits, leaves, and shoots (Buriticá 61
1999; Keith et al. 2006). It is caused by a group of fungi in the genus Pestalotiopsis. The first 62
visible symptoms of guava scab are small brown (coffee-bean colored) spots that develop into 63
corky scabs on fruit surfaces. The scabs develop “heads” that resemble oxidized nails. The lesions 64
coalesce to form large lesions that cover part of the fruit and sink into the flesh, and eventually 65
deform the whole fruit. Leaf symptoms appear as brown lesions that usually show up first on leaf 66
edges and apices. As they expand, lesions become brittle and gray with mycelium growth. 67
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A. F. Solarte et al. Plant Disease 4
Farfán et al. (2006) reported that Pestalotia spp. are widely distributed throughout La Hoya 68
del Río Suárez, Colombia. They also found that in the municipality of Vélez, guava fruits are most 69
susceptible to infection in the transition periods between dry and rainy seasons in March (90.6%) 70
and between rainy and dry in July (93.1%), (Farfán et al. 2006). Guava scab has also been reported 71
in Mexico (Morera and Blanco 2009), India, Australia (Jeewon et al. 2003), and the USA (Hawaii) 72
(Keith et al. 2006). 73
The causal agents of guava scab in Colombia are Pestalotia spp. (Morera and Blanco 2009) 74
and Pestalotiopsis spp. (Keith et al. 2006). However, there have been no efforts to characterize and 75
identify the Pestalotiopsis species attacking the guava crop in Colombia. Additionally, the 76
pathogen’s distribution in Colombia’s principal production areas has not been determined. In 77
general, the guava scab pathogens have been characterized by their morphological features and not 78
by molecular techniques such as multiple-gene analysis. De Notaris (1839) initially described the 79
genus Pestalotia, characterizing it as having a fusiform conidium composed of six cells, and 80
appendages in the apical and basal extremes. Over a century later, Steyaert (1955) divided the 81
genus into three genera according to the number of cells comprising the conidia, specifically, 82
conidia of Pestalotia, Pestalotiopsis, and Truncatella genera possessed 6, 5, and 4 cells, 83
respectively. This last classification is still in current use after more than 60 years 84
(Maharachchikumbura et al. 2011). However, the separation of Pestalotiopsis and Pestalotia into 85
distinct genera remained controversial until 1980, when Sutton (1980) used electron microscopy to 86
examine the development of the cell wall in two species of Pestalotiopsis and in Pestalotia 87
pezizoides. Sutton’s findings supported Steyaert’s classification (Griffiths and Swart 1974a, b). 88
Until the 1990s the taxonomy of Pestalotiopsis and related genera was based on describing 89
conidial characteristics because of the stability of their traits. Specifically the pigmentation of the 90
three medium cells of the conidia are concolorous in Pestalotiopsis and versicolorous in 91
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A. F. Solarte et al. Plant Disease 5
Neopestalotiopsis (Maharachchikumbura et al. 2011, 2012). However, the sole use of conidial 92
characteristics for species identification was controversial because of the wide variability of other 93
morphological characteristics such as colony color, texture, and shape observed for Pestalotiopsis 94
and related genera when grown on culture media (Egger 1995; Hu et al. 2007). 95
Molecular techniques have become the norm for species differentiation and identification. 96
Maharachchikumbura et al. (2012) evaluated 10 groups of primers, seeking the best regions and/or 97
genes on which to perform phylogenetic analyses of multiple, replicable, and reliable genes. The 98
authors succeeded in establishing the internal transcribed spacer (ITS) region and the β-tubulin and 99
translation elongation 1 (tef1) genes as the best regions because of greater amplification through 100
PCR and sharper definition of species limits. 101
Recently, Maharachchikumbura et al. (2014) reclassified Pestalotiopsis into three genera. 102
The sequence analyses of several species based on the large subunit (LSU) gene, together with the 103
results of the 2012 multiple-gene analyses, clearly show three highly diverse lineages. Two new 104
genera—Neopestalotiopsis and Pseudopestalotiopsis—were therefore introduced. The objective of 105
this study was to characterize and assess the genetic diversity of the isolates causing guava scab in 106
Colombia. 107
108
Materials and Methods 109
Sampling, isolation, and storage 110
Samples of infected guava tissues (leaves and fruits) were collected from commercial crops 111
in the Colombian regions of Boyacá, Santander, and Valle del Cauca (Fig 1). These regions 112
produce different guava varieties with different crop management and edaphoclimatic conditions. 113
We obtained infected guava leaf and fruit samples (n=200) from 45 farmers’ fields. 114
Samples had symptoms that included spots that were blackish gray, necrotic, amorphous, and 115
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A. F. Solarte et al. Plant Disease 6
brittle, and were usually located on leaf apices. Or they were round to oval brown, corky lesions 116
that were located on the fruit epidermis. The symptomatic guava varieties that were sampled 117
included Palmira ICA-1, Pera, Coronilla, Regional Roja, and Manzana. 118
Samples (5 mm
2
) were washed first in deionized water for 10 min, followed by 1 min in 119
1% sodium hypochlorite and 1 min in 70% alcohol. Subsequently, samples were washed twice 120
with sterilized distilled water for 1 min. Samples (5 mm
2
) were then dried on sterilized paper 121
towels, and plated onto potato dextrose agar (PDA; Difco Laboratories Inc.) modified with 1 ml of 122
lactic acid at 25% per liter (Merck & Co. Inc.) (five samples per plate). Plates were then incubated 123
at 22°C ± 1 under 12/12h light/darkness for 5 days. 124
To obtain single-spore cultures, we started with 8- to 10-day-old colonies. One acervulus 125
from each colony was picked-up using a fine needle (manufacturer) and washed into a petri dish 126
containing water agar and with one drop of sterilized distilled water. Drops of water carrying 127
acervuli were then spread over the entire surface of the medium, using a sterilized glass spreader. 128
A stereo-microscope was then used to identify one conidium physically separated from the others. 129
A fragment of the culture medium carrying this conidium was transferred to a plate containing 130
fresh PDA medium supplemented with 1 ml of 25% lactic acid per liter (Merck & Co., Inc.). The 131
colonies were incubated at 22ºC ± 1 under 12/12h light/darkness for 1 to 2 days. 132
Morphological characterization 133
We characterized 81 monoconidial isolates from different acervuli of Pestalotiopsis spp. 134
based on morphological features. From the edges of 5-day-old colonies, discs (5 mm in diameter) 135
were transferred to PDA plates and incubated at 22°C under 12/12h light/darkness for 7 days. The 136
morphological characteristics of these cultures were examined 7 days later. Specifically, cultures 137
were examined for colony color, growth rate, presence or absence of acervuli, conidia length and 138
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A. F. Solarte et al. Plant Disease 7
width, apical and basal appendage length and other characteristics as described by Keith et al. 139
(2006) and Maharachchikumbura et al. (2011). To determine
the growth rate, isolates were cultured 140
in 90-mm petri dishes containing PDA in a randomized complete block design with three 141
replications. The experiment was repeated twice. Disks, 5mm in diameter, were cut from the 142
edge of a 5-day-old colony and placed in the center of petri dishes with mycelium in direct contact 143
with PDA medium. The isolates were incubated at 22°C under 12/12h light/darkness for 7 days 144
and radial growth was measured daily. Analysis of variance of characteristics described above was 145
carried out with the Statistical Analysis System (SAS
®
software v. 9.0 Cary, NC, USA)(ANOVA; 146
P < 0.01), and the Ryan-Einot-Gabriel-Welsch (REGW) multiple range test was used to separate 147
means (α = 0.05). 148
To determine the average length, width, and number of appendages of conidia, 20 mature conidia 149
were obtained from each 10-day-old isolate. Conidia were mounted on slides and stained with 150
cotton blue for contrast. Photographs were taken with a Leica microscope at 400x magnification 151
and measurements were made using the ImageJ software (Image Processing and Analysis in Java). 152
The twenty measurements were subjected to an analysis of variance with 20 replications using 153
SAS
®
v. 9.0 software the REGW multiple range test to separate means (α = 0.05). 154
Pathogenicity tests 155
The aggressiveness of the 81 isolates was evaluated by inoculating them onto detached 156
physiologically mature guava fruits (variety Palmira ICA-1). The fruits were disinfected by 157
immersion into 2% sodium hypochlorite for 2 min, then in 70% alcohol for 1 min, and finally 158
rinsing twice in sterilized distilled water for 1 min each time. Fruits were air-dried on sterilized 159
paper towels in a laminar flow chamber for 30 min. 160
The fruits were inoculated as follows: a 5-mm disc of PDA medium with mycelia from a 161
5-day-old culture was placed on a 1.5 mm wound made by removing a piece of fruit epidermis. 162
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A. F. Solarte et al. Plant Disease 8
The disc was then covered with the piece of epidermis and sealed with parafilm. Fruits were then 163
placed on plastic grate inside translucent polypropylene boxes. Water was added to a level below 164
the plastic grate to maintain 100% relative humidity. The boxes were then hermetically sealed and 165
incubated at 22ºC under 12/12 hours of light to darkness for 12 days. 166
Aggressiveness was evaluated as “lesion diameter” (mean diameter of three lesions per 167
day) at 4, 8 and 12 days after inoculation. Each isolate was replicated three times and the 168
experiment was conducted twice. Fruits inoculated in a similar manner with PDA discs with no 169
mycelia served as negative controls. Data were analyzed by ANOVA (P < 0.01), and mean 170
separation tests were conducted using the REGW multiple range test (α = 0.05), using SAS
®
v. 9.0 171
software. 172
Phylogenetic analysis of multiple genes 173
DNA extraction. To extract DNA, we used the methodology reported by Damm et al. 174
(2008, cited in Gramaje et al. 2009). Fragments of 5-day-old fungal mycelium grown on PDA were 175
transferred to 1.5-ml Eppendorf tubes, each containing 200 l of CTAB extraction buffer (0.2 M of 176
Tris pH, 1.4 M of NaCl, 20 mM of EDTA, and 0.2 g/l of CTAB). The mycelium was macerated in 177
600 l of buffer using a sterilized micropestle (Sigma-Aldrich
®
), until pulverized. The tubes were 178
then incubated at 65ºC for 15 min, after which 400 l of chloroform:isoamyl alcohol (24:1) was 179
added to each tube. Tubes were mixed by inverting, and immediately centrifuged at 15,800 g for 5 180
min. 181
The supernatant (ca. 500 l) was transferred to a fresh tube and 600 l of cold isopropanol 182
and 166 l of cold 7.5 M ammonium acetate were added to obtain a final concentration of 2.5 M. 183
The tubes were then incubated at room temperature for 15 min and centrifuged at 15,800 g for 5 184
min. The supernatant was discarded, saving only the pellets. One milliliter of cold 70% ethanol 185
was then added to each tube and centrifuged at 15,800 g for 5 min. The supernatant was discarded 186
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A. F. Solarte et al. Plant Disease 9
and the pellet was dried in a SpeedVac dryer at 37ºC for 50 min. After drying, the pellets were re-187
suspended in TE buffer and 1.5 l of RNase was added to each tube. Tubes were incubated at 37ºC 188
for 30 min. 189
Amplification of the internal transcribed spacer (ITS), β-tubulin, and elongation factor 190
genes. The polymerase chain reaction (PCR) assay was used to amplify the ITS region, and the β-191
tubulin and tef1 genes. The three regions were amplified using primers and amplification 192
conditions that were previously published by (White et al. 1990; Glass and Donaldson 1995; 193
Rehner 2001; Maharachchikumbura et al. 2012). For the PCR amplifications, a 25-l reaction 194
volume was prepared consisting of 10 ng of DNA, a final 1X concentration of Promega GoTaq
®
195
Green Master Mix, 2X, and 0.5 mM of each primer. The reactions were carried out in a PTC-100 196
thermal cycler (MJ Research Inc. Madison, WI). 197
The PCR products were first cleaned by adding 25 l of PEG (20%) and NaCl (2.5 M) 198
solution to each sample. The mixture was homogenized using a vortexer (Scientific Industries, 199
Bohemia, NY), and incubated at room temperature for 15 min. The solution was then centrifuged 200
at 13,000 rpm for 15 min, and the supernatant was discarded. One hundred microliters of 70% 201
ethanol was added to the tube and the solution was centrifuged at 13,000 rpm for 3 min. The 202
ethanol was then carefully removed and each pellet was dried by incubation at 38°C for 30 min. 203
Finally, each DNA pellet was re-suspended in 20 µl of ultrapure distilled water (Invitrogen
TM
,
204
Grand Island,
NY). The amplicons were sent to the DNA Facility of the Iowa State University 205
(Ames, IA) for sequencing. The sequences were aligned to obtain a consensus sequence of the 206
amplified regions for all isolates. This sequence was analyzed using ChromasPro version 1.49 beta 207
and MEGA 5.2 (Kumar et al. 2012). Homologies were sought using the BLAST tool in GenBank 208
(www.nbci.nlm.nih.gov). The reference sequences were downloaded from GenBank species type 209
(holotype, epitype, ex-epitype) and others previously referred to by Maharachchikumbura et al. 210
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A. F. Solarte et al. Plant Disease 10
(2014), Ariyawansa et al. (2015), Liu et al. (2015) and Jayawadena et al. (2016) (Table 1). These 211
sequences were aligned with the sequences obtained from the isolates collected from guava using 212
MEGA 5.2. The aligned sequences were analyzed using Bayesian Inference of Phylogeny, variant 213
Metropolis coupled Markov Chain Monte Carlo (MCMC), and MrBayes software 3.2.1 214
(http://mrbayes.sourceforge.net/download.php) (Ronquist et al. 2012). The best model for 215
substituting nucleotides for the sequences of each primer was determined separately, using the 216
application jModelTest 2.1.4 (http://darwin.uvigo.es/our-software/) (Posada 2008). For the 217
multilocus analysis of the combined ITS region, β-tubulin, and tef1 genetic loci, 30 million 218
generations were run, sampling every 1000 generations and omitting the first 25% of trees 219
generated. From the remaining trees sampled the consensus tree was estimated at a domain of more 220
than 50%. The resulting trees were printed with Fig Tree version 1.4.0 221
(http://tree.bio.ed.ac.uk/software/figtree/) and the layout was made with Adobe Illustrator Cs (V.6.
222
San José, California). 223
224
Results 225
Isolating the fungi 226
We collected 81 isolates from the three regions sampled as follows: 76 Neopestalotiopsis 227
isolates were recovered, with 9 coming from Boyacá, 44 from Santander, and 23 from Valle del 228
Cauca. We also recovered 5 Pestalotiopsis isolates, with 3 from Santander, and 2 from Valle del 229
Cauca. We isolated Neopestalotiopsis spp. from fruits in all phenological stages of development 230
and from mature leaves, while Pestalotiopsis spp. were isolated only from fruits (Fig 2). 231
Neopestalotiopsis isolates came from tissues of productive trees of the four most common varieties 232
(Pera, Manzana, Coronilla, and Regional Roja) found in the sampling zones. In contrast, 233
Pestalotiopsis isolates were found only on cvs. Regional Roja and Pera. 234
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A. F. Solarte et al. Plant Disease 11
235
Morphological characterization---macroscopic 236
No morphological differences were found between Pestalotiopsis and Neopestalotiopsis 237
isolates. The 81 isolates grouped into 9 morphotypes according to colony characteristics (Table 1). 238
The principal descriptors were surface color, texture, mycelium production (high, medium, or 239
low), presence or absence of surface acervuli, and edge type. However, surface color, mycelium 240
production, and texture, were given the most weight because the other characteristics were highly 241
variable. The 81 isolates were distributed throughout the 9 morphotypes as follows: 1 = 33 242
(40.7%); 2 = 16 (19.7%); 7 = 6 (7.4%); 3, 4, 5, and 9 = 5 (6.17%) each; 8 = 4 (4.9%); and 6 = 1 243
(1.2%). 244
Twenty-four (30%) isolates had a high growth rate on artificial culture media, while 31 245
(38%) grew at medium rates. The remaining 26 (32%) isolates grew slowly (Table 2). Moreover, 246
no correlation was found between growth rate on artificial culture medium and colony 247
morphology. 248
249
Morphological characterization—microscopic 250
Microscopic characterization was carried out on 48 isolates that produced fruiting bodies 251
with conidia. Sporulation was low in the remaining 33 isolates. On sub-culturing, these isolates 252
lost morphological characteristics, including the ability to produce fruiting bodies. 253
The minimum and maximum values for conidium length were 19.05 and 29.25 m, 254
respectively, while the minimum and maximum values for conidium width were 4.40 and 255
10.40 m, respectively. The averages ranged from 21.38 to 25.74 m for length and 5.13 to 256
8.34 m for width. 257
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A. F. Solarte et al. Plant Disease 12
The minimum and maximum values for length of apical appendages were 11.54 and 34.8 258
m, respectively, and the minimum and maximum values for length of basal appendages were 3.00 259
and 6.75 m, respectively. For most isolates, appendage number per conidium was constant, with 260
most isolates presenting conidia with three apical and one basal appendages (Table 3). 261
Of the 48 isolates, 46 were Neopestalotiopsis spp. Analysis of their median cells showed 262
that they had versicolored conidia, with two upper median cells being darker than lowest median 263
cell. Forty-five of these isolates had dark-and-pale coffee-colored conidia and one (Vr1eP) 264
presented blackish gray conidia. The two remaining isolates (VTman5 and SSpla) were 265
Pestalotiopsis spp., and had concolorous conidia (Fig 3). 266
The post-hoc test used in ANOVA on conidial length and width data showed significant 267
differences (P = 0.05) for only isolates VTman5 and SSpla, which formed one group, and for the 268
isolate Vr1eP, which formed another group by itself. The characteristics shared by all other isolates 269
were not significantly different. Isolates VTman5 and SSpla also stood out for their long and thin 270
conidia, which were 5.42 and 5.13 m wide, respectively. Their length-to-width ratio was also the 271
highest, and while Vr1eP had a similar length-to-width ratio it had a higher average width 272
(5.94 m). 273
Pathogenicity tests 274
Isolates that did not form lesions were considered as non-pathogenic, and did not differ 275
significantly from the control. Four of the 81 isolates were not pathogenic. Different degrees of 276
aggressiveness were observed among the 77 pathogenic isolates by means of two variables: 277
diameter of the lesion (DL) and area under the disease-progress curve (AUDPC). SAS cluster 278
analysis (Ward’s Minimum Variance Cluster Analysis) separated the isolates into three groups 279
according to their level of aggressiveness (P = 0.05): low, intermediate, and high. 280
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The ANOVA (P < 0.01) for variable DL showed significant differences for aggressiveness, 281
according to the REGW multiple-range test (α = 0.05). The high aggressiveness group was 282
composed of Neopestalotiopsis isolates mainly from Santander and Valle del Cauca; and one 283
(BVayr1) from Boyacá. The most aggressive isolate was SVpo5 from Vélez (Santander), standing 284
out from all the others, including the highly aggressive VRes2, SVpa1, BVayr1, SVsnp8, and 285
VRte1. 286
Fruits attacked by mildly aggressive isolates developed small lesions with few visible 287
acervuli on their surfaces. Intermediately aggressive isolates usually produced a larger quantity of 288
surface acervuli, but small quantities of mycelia. Highly aggressive isolates produced large 289
quantities of mycelia and surface acervuli by 12 days after inoculation. 290
Molecular characterization 291
Phylogenetic analysis. The phylogenetic analysis included reference sequences from 52 292
Pestalotiopsis and 27 Neopestalotiopsis species, all 79 epitypified, and 13 Neopestalotiopsis spp. 293
published in Maharachchikumbura et al. (2014), Ariyawansa et al. (2015), Liu et al. (2015), and 294
Jayawardena et al. (2016). The sequences from this study are available in GenBank as accession 295
numbers KR493595 to KR493664. 296
Sequence analysis enabled the separation of isolates into two genera: Pestalotiopsis (Fig 4) 297
and Neopestalotiopsis (Fig 5). Moreover, this analysis enabled us to establish that under our 298
study’s conditions, the Neopestalotiopsis isolates were more numerous (63) than the Pestalotiopsis 299
isolates (4). The phylogenetic analysis therefore presents them in separate Cladograms. 300
The four Pestalotiopsis are grouped into two clades (Fig 4); the first two (VTsin2 and 301
SSpla) formed one clade, Pestalotiopsis sp. 1; and the other two isolates (SVsnp4 and VTman5) 302
formed a separate clade with P. australasiae. 303
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For the Neopestalotiopsis group shown in Fig 5, the 63 isolates in this study were 304
distributed across 11 well-defined clades. Of the 63 isolates, 40 were grouped into two clades, 305
Neopestalotiopsis sp. 1 and Neopestalotiopsis sp. 3, with no reference sequences being present. 306
Clade Neopestalotiopsis sp. 1 was more homogeneous. 307
Some sequences of Neopestalotiopsis spp. were closely related to reference species. For 308
example, BVayr1 and SPugra5 formed a clade with N. surinamensis. Likewise, VR1ep formed a 309
clade with two sequences of N. foedans; VUgu2 with two sequences that have not yet been 310
determined; and SVsnp10, SVsnp12, SPugra3, and SVsnp8 formed a clade with N. egyptiaca and 311
VTman2, showing high sequence similarity (98.6%) (Fig 5). 312
Although no relationship was observed among the isolates based on geographical origin, 313
most of the clades were formed by isolates from Santander and Valle de Cauca. Nevertheless, 314
some clades and subclades were grouped isolates from specific regions. For example, clade 315
Neopestalotiopsis sp. 1 has a subclade of three isolates (SSre1, SSre2, and SSre4) that came from 316
the same region in Santander; clade Neopestalotiopsis sp. 2 is formed by three isolates from 317
Boyacá; and clade Neopestalotiopsis sp. 8 is formed by two isolates from Santander. 318
Beyond these similarities, we could not determine the species of most of the isolates 319
because, there are only a few nucleotides of difference between the sequences of the species in the 320
genetic loci we studied. However, both Pestalotiopsis and Neopestalotiopsis are clearly associated 321
with the disease in Colombia, and the isolates showed substantial diversity. 322
323
Discussion 324
This is the first study documenting the genetic relationships between isolates of 325
Neopestalotiopsis and Pestalotiopsis causing guava scab. Molecular and morphological 326
characterization along with virulence assays demonstrated that guava scab was caused by more 327
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A. F. Solarte et al. Plant Disease 15
than one species (Neopestalotiopsis and Pestalotiopsis). Selection due to geographic location was 328
not an important factor determining genetic structure of clades. There was no correlation among 329
origin, morphology, pathogenicity, and molecular clades based on multiple gene analysis of the 330
isolates in Colombia (Table 1). 331
In this study, 77 of 81 isolates recovered from guava scab lesions were pathogenic to 332
detached guava fruits. Virulence assays on fruits of guava cultivar Pera1 also revealed that isolates 333
SVpo5, VRes2, SVpa1 and BVayr1 were more aggressive than isolates SVsnp5, Svsnp11 and 334
SVpa8. In addition, isolates VUgu3, VTman4, SVpa2 and BVayr2 were not pathogenic. The 335
aggressive strains produce symptoms of guava scab on fruit tissues. 336
We observed the same variability in colony color (white, pale buff, pale saffron, or pale 337
olive) and production of acervuli as Keith et al. (2006). Also conidium length was similar to that 338
reported by Keith et al. (2006). In other studies, conidium length was reported as being as short as 339
15 µm to as long as 35 µm (Hu et al. 2007; Maharachchikumbura et al. 2013, 2014). 340
Most of the isolates recovered from guava scab lesions exhibited variability in the 341
morphological characteristics evaluated. When sub-cultured, the isolates changed one or more 342
colony characteristics. This was previously reported by Hu et al. (2007), who indicated that 343
Pestalotiopsis spp. colony morphology is not stable under sub-culturing (e.g., for color, growth 344
rate, and texture). Indeed, a single isolate was sometimes seen as having two different 345
morphologies. Jeewon et al. (2003), Tejesvi et al. (2007), and Maharachchikumbura et al. (2011) 346
suggest that colony morphology characteristics are plastic and variable. In contrast, conidial 347
characteristics are more stable, especially the length, width, and color of median cells. These traits 348
were more useful for differentiating isolates, enabling statistical analyses to separate the four 349
Pestalotiopsis species evaluated (VTsin2, SSpla, SVsnp4, and VTman5) from those of the 350
Neopestalotiopsis species. However, it was impossible to separate or group isolates, especially 351
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A. F. Solarte et al. Plant Disease 16
those with unreliable traits. This situation made it necessary to use phylogenetic analysis to better 352
understand the other traits and variables analyzed in our study. Maharachchikumbura et al. (2012) 353
came to the same conclusion. 354
Phylogenetic analyses revealed genetic variability among the guava scab isolates, 355
particularly those of the Neopestalotiopsis species. The possibility of finding more than one 356
species causing guava scab was already reported by Keith et al. (2006), who found 5 Pestalotiopsis 357
species (P. microspora, P. clavispora, P. disseminata, P. neglecta, and Pestalotiopsis sp. GJ-1) 358
among 23 isolates collected from different guava varieties in Hawaii. Unfortunately, none of these 359
species is epitypified and therefore could not be included in our study. 360
Phylogenetic analysis also revealed close relationships between some reference sequences 361
and isolates in our study. For Pestalotioipsis, isolates SVsnp4, VTman5 were related to 362
P. australasiae; and for Neopestalotiopsis, BVayr1, SPugra5 were related to N. surinamensis. 363
Additionally, VTman2 was closely related to N. egyptiaca; Vrlep was closely related to N. foedans 364
and VUgu2 was related to Neopestalotiopsis sp. CBS 360.61, and Neopestalotiopsis sp. CBS 365
162.42. Despite this, the expertise of a taxonomist specializing in these genera should be consulted 366
since sequence similarity is not sufficient to identify the species. The foregoing is evident in the 367
studies by Maharachchikumbura et al. (2012, 2014), where two species were differentiated by only 368
one pair of nucleotides. For example, in the 2014 study, N. honoluluana and N. zimbabwana 369
differed by only six nucleotides, but were defined as two species. In contrast, in the 2012 study, 370
one of the N. foedans isolates differed by six nucleotides, but was considered to be the same 371
species as the other two N. foedans isolates evaluated. 372
This situation shows the importance of combining morphological traits with phylogenetic 373
analysis when determining a new species. In our study, we could not reach this level of depth, as 374
our objective was phytopathological rather than taxonomic. Nevertheless, our data showed that the 375
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A. F. Solarte et al. Plant Disease 17
guava scab in Colombia is caused by a complex of species of the genera Pestalotiopsis and 376
Neopestalotiopsis. These results agree with those of Keith et al. (2006), taking into account that in 377
their study, the Pestalotiopsis and Neopestalotiopsis genera were classified as one genus. 378
The isolates belonging to 6 of the 11 clades (Neopestalotiopsis spp. 1, 2, 3, 5, 6, and 11) 379
could be defined as new species (S.S.N. Maharachchikumbura, personal communication). It is 380
even possible that some clades may include more than one species, for example, clades 381
Neopestalotiopsis spp. 1, 3, and 5 (Fig 5).Our findings represent an important contribution to the 382
etiology and epidemiology of guava scab in Colombia. We provide evidence for diversity in both 383
the Neopestalotiopsis and Pestalotiopsis spp. associated with the disease in guava. Our study 384
facilitates the development of future epidemiological research in guava and other crops. It offers 385
information relevant to the design of management strategies, such as appropriate chemistries and 386
crop rotation, and to the evaluation of resistance in Psidium germplasm to the different 387
Neopestalotiopsis and Pestalotiopsis isolates. 388
389
Acknowledgments 390
This work was financed by the OPEC Fund for International Development (OFID) (2012 to 391
2013). 392
We thank Nedy Ramírez who participated in field collection, Napoleón and David Vargas 393
for managing and collaborating with field collections in Vélez; and Juan Cuasquer for data 394
analysis. We also thank the following for their assistance: Lederson Gañan, Germán Ceballos, Juan 395
Manuel Pardo, Michael Latorre, Zulma Zamora, Viviana Domínguez, and Alvaro Soler. 396
397
398
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A. F. Solarte et al. Plant Disease 18
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A. F. Solarte et al. Plant Disease 22
Table 1. Details of Pestalotiopsis and Neopestalotiopsis isolates obtained from guava fruits and leaves collected from Boyacá,
494
Santander and Valle del Cauca in Colombia included in this study
495
Species Isolate code Origen Source Genotype
Growth rate
a
(mm)
Neopestalotiopsis SSbu1 San Benito, Santander Fruit Roja 16,9
Neopestalotiopsis SVsnp8 Vélez, Santander Leaf Roja 16,7
Neopestalotiopsis
VTman1 Toro, Valle Fruit Pera 16,7
Neopestalotiopsis
VUve3 La Unión, Valle Fruit Pera 16,5
Neopestalotiopsis
SVsnp3 Vélez, Santander Leaf Roja 15,6
Neopestalotiopsis
SVpa1 Vélez, Santander Leaf Roja 15,6
Neopestalotiopsis
BVayr1 Pauna, Boyacá Fruit Roja 15,4
Neopestalotiopsis
VTman2 Toro, Valle Fruit Pera 15,3
Neopestalotiopsis
VTman3 Toro, Valle Leaf Pera 15,3
Neopestalotiopsis
SBma1 Barbosa, Santander Fruit Roja 15,1
Neopestalotiopsis
SPugra2 P. Nacional, Santander Fruit Roja 15,1
Neopestalotiopsis
SVpo3 Vélez, Santander Fruit Roja 15
Neopestalotiopsis
SVsnp11 Vélez, Santander Fruit Roja 15
Neopestalotiopsis
VUve2 La Unión, Valle Fruit Coronilla 14,9
Neopestalotiopsis
SBac2 Barbosa, Santander Leaf Roja 14,9
Neopestalotiopsis
VRes2 Roldanillo, Valle Fruit Manzana 14,9
Neopestalotiopsis
SPugra1 P. Nacional, Santander Fruit Roja 14,8
Neopestalotiopsis
SVpa4 Vélez, Santander Fruit Roja 14,7
Neopestalotiopsis
SVpo7 Vélez, Santander Leaf Roja 14,7
Neopestalotiopsis
SPugra5 P. Nacional, Santander Fruit Roja 14,6
Neopestalotiopsis
BPpa2 Pauna, Boyacá Fruit Roja 14,5
Neopestalotiopsis
SVpo5 Vélez, Santander Leaf Roja 14,4
Pestalotiopsis
VTsin3 Toro, Valle Leaf Pera 14,4
Neopestalotiopsis
VRte1 Roldanillo, Valle Leaf Pera 14,4
Neopestalotiopsis
SSbu2 San Benito, Santander Fruit Roja 14,1
Neopestalotiopsis
SSre5 San Benito, Santander Leaf Roja 14,0
Neopestalotiopsis
SVsnp12 Vélez, Santander Fruit Roja 13,8
Neopestalotiopsis
SVpa3 Vélez, Santander Fruit Roja 13,7
Neopestalotiopsis
SVsnp2 Vélez, Santander Fruit Roja 13,7
Neopestalotiopsis
SVsnp7 Vélez, Santander Fruit Roja 13,7
Neopestalotiopsis
BPva1 Pauna, Boyacá Fruit Roja 13,6
Neopestalotiopsis
SVsnp1 Vélez, Santander Leaf Roja 13,6
Neopestalotiopsis
VRes4 Roldanillo, Valle Fruit Manzana 13,5
Pestalotiopsis
VTman5 Toro, Valle Fruit Pera 13,5
Neopestalotiopsis
BTca2 Tununguá, Boyacá Fruit Roja 13,3
Neopestalotiopsis
SVpo2 Vélez, Santander Fruit Roja 13,3
Neopestalotiopsis
VUgy La Unión, Valle Fruit Pera 13,2
Neopestalotiopsis
SVsnp9 Vélez, Santander Fruit Roja 13,1
Pestalotiopsis
VTsin2 Toro, Valle Leaf Pera 13,1
Neopestalotiopsis
SVpa8 Vélez, Santander Fruit Roja 12,9
Neopestalotiopsis
VUve1 La Unión, Valle Fruit Pera 12,8
Neopestalotiopsis
BPca2 Pauna, Boyacá Fruit Roja 12,7
Neopestalotiopsis
SSre3 San Benito, Santander Leaf Roja 12,7
Neopestalotiopsis
SSbu3 San Benito, Santander Fruit Roja 12,7
Neopestalotiopsis
SSre2 San Benito, Santander Leaf Roja 12,7
Neopestalotiopsis
VUgu1 La Unión, Valle Fruit Pera 12,6
Neopestalotiopsis
SVpo1 Vélez, Santander Fruit Roja 12,5
Neopestalotiopsis
SVsnp13 Vélez, Santander Fruit Roja 12,5
Neopestalotiopsis
VTman6 Toro, Valle Leaf Pera 12,5
Neopestalotiopsis
SVpo6 Vélez, Santander Fruit Roja 12,3
Neopestalotiopsis
SVsnp6 Vélez, Santander Fruit Roja 12,3
Neopestalotiopsis
VTsin1 Toro, Valle Leaf Pera 12,2
Neopestalotiopsis
SVpa7 Vélez, Santander Fruit Roja 12,2
Neopestalotiopsis
SVsnp5 Vélez, Santander Leaf Roja 12,1
Neopestalotiopsis
SVpo2-2 Vélez, Santander Fruit Roja 12,1
Neopestalotiopsis
BPca1 Pauna, Boyacá Fruit Roja 11,7
Neopestalotiopsis
SVpa6 Vélez, Santander Fruit Roja 11,7
Neopestalotiopsis
SBac1 Barbosa, Santander Leaf Roja 11,7
Neopestalotiopsis
SBma3 Barbosa, Santander Fruit Roja 11,6
Pestalotiopsis
SSpla San Benito, Santander Fruit Roja 11,6
Neopestalotiopsis
VRes1 Roldanillo, Valle Fruit Manzana 11,5
Neopestalotiopsis
VUve4 La Unión, Valle Fruit Pera 11,5
Neopestalotiopsis
SVpa5 Vélez, Santander Fruit Roja 11,5
Neopestalotiopsis
SPugra4 P. Nacional, Santander Fruit Roja 11,3
Neopestalotiopsis
VUgu2 La Unión, Valle Fruit Pera 11,1
Neopestalotiopsis
SPugra3 P. Nacional, Santander Fruit Roja 11
Neopestalotiopsis
SVpo4 Vélez, Santander Fruit Roja 11
Neopestalotiopsis
BTca1 Tununguá, Boyacá Fruit Roja 11
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A. F. Solarte et al. Plant Disease 23
Neopestalotiopsis
VRes3 Roldanillo, Valle Fruit Manzana 10,8
Neopestalotiopsis
VRlep Roldanillo, Valle Fruit Pera 10,8
Pestalotiopsis
SVsnp4 Vélez, Santander Fruit Roja 10,5
Neopestalotiopsis
BPpa1 Pauna, Boyacá Fruit Roja 9,7
Neopestalotiopsis
SVsnp10 Vélez, Santander Leaf Roja 8,9
Neopestalotiopsis
VTman4 Toro, Valle Leaf Pera 8,5
Neopestalotiopsis
SBma2 Barbosa, Santander Fruit Roja 8,4
Neopestalotiopsis
BVayr2 Pauna, Boyacá Fruit Roja 8,3
Neopestalotiopsis
VTpo Roldanillo, Valle Fruit Pera 8,1
Neopestalotiopsis
SVpa2 Vélez, Santander Fruit Roja 8,1
Neopestalotiopsis
SSre4 San Benito, Santander Fruit Roja 7,9
Neopestalotiopsis
VUgu3 La Unión, Valle Fruit Pera 7,2
Neopestalotiopsis
SSre1 San Benito, Santander Leaf Roja 5,8
496
a
Rate of growth on potato dextrose agar: high (14,4 to 16,9); medium (12,1 to 14,1); low (5,8 to 11,7)
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
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A. F. Solarte et al. Plant Disease 24
Table 2. Details of isolates representing species in the phylogenetic clades of Pestalotiopsis and Neopestalotiopsis used in this
512
study
a
513
Species
GenBank accesión numberb
Host/Substrate
Location
Neopestalotiopsis aotearoa CBS 367.54; ATCC 11763; QM 381 Canvas New Zealand
N. asiatica MFLUCC 12-0286; NN0476380 Unidentified tree China
N. australis CBS 114159; STE-U 3017 Telopea sp. Australia
N. chrysea MFLUCC 12-0261; NN042855 Dead leaves China
N. chrysea MFLUCC 12-0262; NN047037 Dead plant China
N. clavispora CBS 447.73 Decaying wood Sri Lanka
N. clavispora MFLUCC 12-0280; NN043011 Magnolia sp. China
N. clavispora MFLUCC 12-0281; NN043133 Magnolia sp. China
N. cubana CBS 600.96; INIFAT C96/44-4 Leaf litter Cuba
N. ellipsospora CBS 115113; HKUCC 9136 Ardisia crenata Hong Kong
N. ellipsospora MFLUCC 12-0283 Dead plant materials China
N. eucalypticola CBS 264.37; BBA 5300 Eucalyptus globulus —
N. egyptiaca CBS 140162 Mangifera indica Egypt
N. foedans CGMCC 3.9123 Mangrove plant China
N. foedans CGMCC 3.9178 Neodypsis decaryi China
N. foedans CGMCC 3.9202 Calliandra haematocephala China
N. formicarum CBS 115.83 Plant debris Cuba
N. formicarum CBS 362.72 Dead Formicidae (ant) Ghana
N. honoluluana CBS 111535; STE-U 2078 Telopea sp. USA: Hawaii
N. honoluluana CBS 114495; STE-U 2076 Telopea sp. USA: Hawaii
N. iraniensis CBS 137768 Fragaria×ananassa Iran
N. javaensis CBS 257.31 Cocos nucifera Indonesia: Java
N. magna MFLUCC 12-652; ICMP 20011 Pteridium sp. France
N. mesopotamica CBS 299.74 Eucalyptus sp. Turkey
N. mesopotamica CBS 336.86 Pinus brutia Iraq
N. natalensis CBS 138.41 Acacia mollissima South Africa
N. piceana CBS 225.30 Mangifera indica —
N. piceana CBS 254.32 Cocos nucifera Indonesia: Sulawesi
N. piceana CBS 394.48 Picea sp. UK
N. protearum CBS 114178; STE-U 1765* Leucospermum cuneiforme Zimbabwe
N. rosae CBS 101057 Rosa sp. New Zealand
N. rosae CBS 124745 Paeonia suffruticosa USA
N. samarangensis CBS 115451; HKUCC 9095 Unidentified tree Hong Kong
N. saprophytica CBS 115452; HKUCC 8684 Litsea rotundifolia Hong Kong
Neopestalotiopsis sp. CBS 233.79 Crotalaria juncea India
Neopestalotiopsis sp. CBS 110.20 — —
Neopestalotiopsis sp. CBS 177.25 Dalbergia sp. —
Neopestalotiopsis sp. CBS 274.29 Cocos nucifera Indonesia: Java
Neopestalotiopsis sp. CBS 322.76 Camellia sp. France
Neopestalotiopsis sp. CBS 664.94 Cocos nucifera Netherlands
Neopestalotiopsis sp. CBS 164.42 Dune sand France
Neopestalotiopsis sp. CBS 360.61 Cinchona sp. Guinea
Neopestalotiopsis sp. CBS 119.75 Achras sapota India
Neopestalotiopsis sp. CBS 266.80 Vitis vinifera India
Neopestalotiopsis sp. CBS 266.37; BBA 5087; IMI 083708 Erica sp. Germany
Neopestalotiopsis sp. CBS 323.76 Erica gracilis France
Neopestalotiopsis sp. CBS 361.61 Cissus sp. Netherlands
N. steyaertii IMI 192475 Eucalyptus viminalis Australia
N. surinamensis CBS 111494; STE-U 1779 Protea eximia Zimbabwe
N. umbrinospora MFLUCC 12-0285; NN042986 Unidentified plant China
N. vitis MFLUCC 15-1265 Vitis vinifera China
N. zimbabwana CBS 111495; STE-U 1777 Leucospermum cunciforme Zimbabwe
Pestalotiopsis adusta ICMP 6088 On refrigerator door PVC gasket Fiji
P. anacardiacearum IFRDCC 2397 Mangifera indica China
P. arceuthobii CBS 434.65 Arceuthobium campylopodum USA
P. arengae CBS 331.92 Arenga undulatifolia Singapore
P. australasiae CBS 114126; STE-U 2896 Knightia sp. New Zealand
P. australis CBS 111503; STE-U 1770 Protea neriifolia × susannae South Africa
P. biciliata CBS 124463 Platanus × hispanica Slovakia
P. biciliata CBS 236.38 Paeonia sp. Proteaceae
P. biciliata CBS 790.68 Taxus baccata Taxaceae
P. brassicae CBS 170.26 Brassica napus New Zealand
P. camelliae CBS 443.62 Camellia sinensis Turkey
P. chamaeropis CBS 113604; STE-U 3078 — —
P. clavata MFLUCC 12-0268; NN0471340 Buxus sp. China
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A. F. Solarte et al. Plant Disease 25
P. colombiensis CBS 118553; CPC 10969 Eucalyptus eurograndis Colombia
P. diploclisiae CBS 115449; HKUCC 9103 Psychotria tutcheri Hong Kong
P. diversiseta MFLUCC 12-0287; NN0472610 Rhododendron sp. China
P. ericacearum IFRDCC 2439 Rhododendron delavayi China
P. furcata MFLUCC 12-0054; CPC 20280 Camellia sinensis Thailand
P. gaultheria IFRD 411-014 Gaultheria forrestii China
P. grevilleae CBS 114127; STE-U 2919 Grevillea sp. Australia
P. hawaiiensis CBS 114491; STE-U 2215 Leucospermum sp. USA: Hawaii
P. hollandica CBS 265.33 Sciadopitys verticillata Netherlands
P. humus CBS 115450; HKUCC 9100 Ilex cinerea Hong Kong
P. inflexa MFLUCC 12-0270; NN0470980 Unidentified tree China
P. intermedia MFLUCC 12-0259; NN0476420 Unidentified tree China
P. jesteri CBS 109350 = MONT 6M-B-3 Fragraea bodenii Papua New Guinea
P. kenyana CBS 442.67 Coffea sp. Kenya
P. knightiae CBS 111963; STE-U 2905 Knightia sp. New Zealand
P. linearis MFLUCC 12-0271; NN0471900 Trachelospermum sp. China
P. malayana CBS 102220 Macaranga triloba Malaysia
P. monochaeta CBS 144.97 Quercus robur Netherlands
P. montelica MFLUCC12-0279 — Thailand
P. novae-hollandiae CBS 130973 Banksia grandis Australia
P. oryzae CBS 111522; STE-U 2083 Telopea sp. USA: Hawaii
P. papuana CBS 331.96 Coastal soil Papua New Guinea
P. parva CBS 265.37; BBA 2820 Delonix regia —
P. portugalica CBS 393.48 — Portugal
P. rhododendri IFRDCC 2399 Rhododendron sinogrande China
P. rosea MFLUCC 12-0258; NN0471350 Pinus sp. China
P. shorea MFLUCC12-0314 Shorea obtusa Thailand
P. scoparia CBS 176.25 Chamaecyparis sp. —
P. spathulata CBS 356.86 Gevuina avellana Chile
P. telopeae CBS 113606; STE-U 3082 Telopea sp. Australia
P. trachicarpicola IFRDCC 2403 Podocarpus macrophyllus China
P. unicolor MFLUCC 12-0275; NN0473080 Unidentified tree China
P. verruculosa MFLUCC 12-0274; NN0473090 Rhododendron sp. China
514
a
Phylogenetic clades of genus Pestalotiops is and Neopestalotiopsis as decribed by Maharachchikumbura et al. (2014), Ariyawansa et al. (2015), Liu et al. (2015) and
515
Jayawadena et al. (2016).
516
b
CBS: culture collection of the Centralbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CGMCC: China General Microbiological
517
Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing; ICMP: International Collection of Microorganisms from Plants,
518
Auckland, New Zealand; IFRDCC: International Fungal Research & Development Centre Culture Collection, China; MFLUCC: Mae Fah Luang University Culture
519
Collection, Chiang Rai, Thailand and IMI: International Micologycal Institute, England.
520
521
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A. F. Solarte et al. Plant Disease 26
Table
3
.
Morphological, cultural, and pathogenic characteristics of 65 Neopestalotiopsis and 4 Pestalotiopsis isolates obtained from guava and grouped according to genetic
diversity clusters, as identified through multilocus analysis
Multilocus
cladev
Infected
tissue Colony typew Genus/species
Origin
Conidium size
AUDPCx averagey
(max–min)
Average
growth
(mm/day)z
Average
length
(µm)
Average
width
(µm)
Length-
to-width
ratioy
Clade 1
p
(2)
Fruit 1 Pestalotiopsis Santander, Valle del Cauca 24.7 5.4 4.5 a 27.7 (36.6–18.8) ab 11.9
Clade 2
p
(2)
Fruit, leaf 1, 5 P. australasiae Santander, Valle del Cauca 22.9 5.1 4.4 a 28.6 (36.5–20.7) ab 12.3
Clade 1
(19)
Fruit, leaf 1, 2, 3, 4, 5,
7, 8, 9
Neopestalotiopsis Santander, Valle del Cauca 23.5 6.6 3.5 cd 27.5 (7.5–75.7) abc 12.7
Clade
2
(3)
Fruit 1, 3 Neopestalotiopsis Boyacá 23.1 6.2 3.7 cd 31.6 (24.9–39.4) b 15.3
Clade 3
(21)
Fruit, leaf 1, 2, 3, 4, 5, 6,
7, 8, 9
Neopestalotiopsis Boyacá, Santander,
Valle del Cauca
23.4 7.1 3.2 ef 38.1 (0–99.2) abc 13.5
Clade 4
(2)
Fruit 1, 2 N. surinamensis Boyacá, Santander 22.7 6.6 3.4 de 62.6 (58.8–66.4) a 15.0
Clade 5
(8)
Fruit 1, 2, 3, 9 Neopestalotiopsis Santander, Valle del Cauca 22.8 6.3 3.5 cd 24.8 (3.5–54) abc 12.3
Clade 6
(3)
Fruit, leaf 1 Neopestalotiopsis Santander, Valle del Cauca 22.5 6.5 3.4 de 4.6 (0–7) c 13.4
Clade 7
(1)
Fruit 2 N. egyptiaca Valle del Cauca - - - 11.8 c 10.8
Clade 8
(4) Fruit, leaf 2, 3, 4 Neopestalotiopsis Santander, Valle del Cauca 23.5 6.1 3.8 cd 48.0 (70.4–15.9) ac 12.5
Clade 9
(1)
Fruit 1 N. foedans Valle del Cauca 24.5 5.9 4.1 b 11.8 c 10.8
Clade 10
(1)
Fruit 8 Neopestalotiopsis Valle del Cauca 21.9 6.8 3.2 ef 11.9 c 11.1
Clade
11
(2)
leaf 1, 7 Neopestalotiopsis Santander, Valle del Cauca 24.3 8.0 3.0 f 34.0 (28.7–39.3) ab 14.5
A
v
Values in parentheses show the number of isolates in each clade.
w
Nine colony morphotypes, where colony:
1 = bright-white colony, cottony texture, intermediate mycelium density, acervuli in the middle, regular edges
2 = bone-white colony, velvety texture, high mycelium density, no acervuli, regular edges
3 = bright-white colony, spiky cottony texture, low mycelium density, scattered acervuli, irregular edges
4 = white colony, powdery spiky texture, intermediate mycelium density, acervuli in the middle, irregular edges
5 = white to bright-yellow colony, powdery texture, low mycelium density, scattered acervuli, regular edges
6 = white colony, powdery texture, low mycelium density, scattered acervuli, regular edges
7 = white colony, powdery texture, intermediate mycelium density, abundant acervuli, regular edges
8 = white colony, powdery texture, low mycelium density, scattered acervuli, irregular edges
9 = black colony, oily texture, low mycelium density, scattered acervuli, irregular edges.
x
AUDPC refers to area under the disease-progress curve.
y
Mean difference between each group is significant at the 0.05 level; values with the same letters in the column do not differ significantly, according to the REGW multiple-
range test.
z
Rate of growth on potato dextrose agar, with no significant differences between clades.
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A. F. Solarte et al. Plant Disease 27
Figure captions
Fig 1. Sampling areas from which tissues of guava (Psidium guajava L.), infested with scab
were obtained.
Fig 2. Symptoms of guava scab: A, leaf, B, immature fruit, and C, mature fruit.
Fig 3. Typical conidia of A, Pestalotiopsis spp., showing concolorous median cells; and
B, Neopestalotiopsis spp., showing versicolored median cells. (Photos taken at 100X.)
Fig 4. Phylogenetic consensus tree based on Bayesian inference, illustrating the relationships
within the Pestalotiopsis epitypified species and of isolates obtained from guava. The tree was
built using concatenated sequences of the genes ITS, β-tubulin, and tef1, and run for 1 x 10
7
generations, each with a separate model of DNA evolution. Neopestalotiopsis saprophyta
(NN047136) was used as the outgroup.
Fig 5. Phylogenetic consensus tree based on Bayesian inference, illustrating the relationships
within the Neopestalotiopsis epitypified species and of isolates obtained from guava. The tree
was built using concatenated sequences of the genes ITS, β-tubulin, and tef1, and run for 1 x 10
7
generations, each with a separate model of DNA evolution. Pseudopestalotiopsis theae
(NN047136) was used as the outroup.
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Sampling areas from which tissues of guava (Psidium guajava L.), infested with scab disease, were obtained
1164x899mm (72 x 72 DPI)
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Symptoms of scab disease in guava: A, leaf, B, immature fruit, and C, mature fruit.
172x40mm (150 x 150 DPI)
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Typical conidia of A, Pestalotiopsis spp., showing concolorous median cells; and B, Neopestalotiopsis
spp., showing versicolored median cells. (Photos taken at 100X.)
131x46mm (150 x 150 DPI)
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Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-01-17-0068-RE • posted 08/16/2017
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Phylogenetic consensus tree based on Bayesian inference, illustrating the relationships within the
Pestalotiopsis epitypified species and of isolates obtained from guava. The tree was built using concatenated
sequences of the genes ITS, β-tubulin, and tef1, and run for 1 x 107 generations, each with a separate
model of DNA evolution. Neopestalotiopsis saprophyta (NN047136) was used as the outgroup
861x1187mm (96 x 96 DPI)
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Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-01-17-0068-RE • posted 08/16/2017
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Phylogenetic consensus tree based on Bayesian inference, illustrating the relationships within the
Neopestalotiopsis epitypified species and of isolates obtained from guava. The tree was built using
concatenated sequences of the genes ITS, β-
tubulin, and tef1, and run for 1 x 107 generations, each with a
separate model of DNA evolution. Pseudopestalotiopsis theae (NN047136) was used as the outgroup
777x1534mm (96 x 96 DPI)
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