Botanical Studies (2012) 53: 467-478.
PHYSIOLOGY
New Botryosphaeriaceae fruit rot of mango in Taiwan: identification and pathogenicity
Hui-Fang NI1'2, Hong-Ren YANG1, Ruey-Shyang CHEN3, Ruey-Fen LIOU2'*, and Ting-Hsuan
HUNG2'*
1Department of Plant Protection, Chiayi Agricultural Experiment Station, Taiwan Agricultural Research Institute, Chiayi,Taiwan
2Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan
3Department of Biochemical Science and Technology, National Chiayi University, Chiayi, Taiwan
(Received February 21, 2012; Accepted May 29, 2012)
ABSTRACT.
Mango is an important fruit crop in Taiwan. Recently, severe fruit rot disease was found fre-
quently on harvested mango fruits. To monitor the incidence of disease and to characterize the causal agent, we performed a field survey in the major mango-producing areas of southern Taiwan, including Guntain, Fan-shan, and Yujing, during 2009-2011. The results showed a disease incidence ranging from 18.7% to 58.1%, with those of Guntain significantly greater than the incidence found in Yujing and Fanshan. Based on morpho­logical characteristics and nucleotide sequences of the internal transcribed spacer (ITS), β-tubulin gene (TUB) and elongation factor 1 -alpha (EF1-α) gene, we identified four Botryosphaeriaceae species, including Fusi-coccum aesculi, Neofusicoccum mangiferae, N. parvum, and Lasiodiplodia theobromae. Pathogenicity tests indicated that all of these fungal species were pathogenic to harvested mango fruits, and L. theobromae was the most aggressive pathogen. Moreover, when attached, immature mango fruits were inoculated with conidia of Botryosphaeriaceae species, disease symptoms characteristic of fruit rot appeared on the fruits after harvest and ripening. These findings indicated that L. theobromae, F. aesculi, N. mangiferae, and N. parvum were all causal agents of the new fruit rot of mango. Furthermore, their conidia may serve as important sources of in-ocula causing fruit rot disease in mango orchards.
Keywords:
Botryosphaeriaceae; Fruit rot; Mango.
INTRODUCTION
Phomopsis mangiferae (Ploetz, 1994; Ko et al., 2009) are usually considered to be the most severe postharvest disease of mango worldwide. However, we have recently found that many harvested mango fruits displayed brown soft lesions on the body surface of ripe mango fruit, rather than at the pedicel end, which indicated that fruit rot could be another serious postharvest disease in Taiwan.
Mango (Mangifera indica) is an economically im­portant fruit crop in Taiwan. According to Agricultural Statistics Yearbook 2010 (http://www.coa.gov.tw/view.php?catid=23771), the total area of mango cultivation in Taiwan was 16,796 ha, which led to the production of 135,293 metric tons of mango fruits, with a yearly value over 157 million US dollars. The main area for mango cul­tivation is located in the southern part of Taiwan, including Tainan, Kaohsiung, and Pingtung.
Botryosphaeriaceae species are known to occur world­wide, causing dieback, cankers, shoot blights, leaf spot, gummosis, and fruit rots in a wide range of plant hosts which play important roles in agriculture and forestry (Phillips, 2002; van Niekerk et al., 2004; Slippers et al., 2005; Damn et al., 2007; de Macedo and Barreto, 2008; Marincowitz et al., 2008; Javier-Alva et al., 2009; Yu et al., 2009; Wang et al., 2011).
Postharvest diseases, which cause serious problems during storage and transportation of mango fruits, are the major factors that limit the thriving mango industry. Both anthracnose disease caused by Colletotrichum gloeospo-rioides (Ploetz, 1994; Yang and Leu, 1988; Arauz, 2000) and stem-end rot caused by Lasiodiplodia theobromae (Liao, 1975; Johnson and Cooke, 1991; Ploetz, 1994) or
In Taiwan, the first plant pathogenic species of Botry-osphaeriaceae that caused mango stem-end rot was re­ported by Liao (1975). He isolated the pathogen Diplodia natalensis (syns. Lasiodiplodia theobromae and Botryodi-plodia theobromae (teleomorph: Botryosphaeria rhodina)) and confirmed its pathogenicity. Recent studies from several laboratories demonstrated that a complex of Botry­osphaeriaceae pathogens (Slippers et al., 2005; de Oliveira
*Corresponding authors: E-mail: rfliou@ntu.edu.tw; Fax: 886-2-23620271; Tel: 886-2-33665208 (Ruey-Fen LIOU); E-mail: thhung@ntu.edu.tw; Fax: 886-2-23636490; Tel: 886-2-33664600 (Ting-Hsuan HUNG).
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Costa et al., 2010), including L. theobromae, N. mangifer­ae, Neofusicoccum parvum and Fusicoccum aesculi, are associated with stem-end rot of mango. Take for example Australia, where F. aesculi (= B. dothidea), N. mangiferae, N. parvum, L. theobromae, and Fusicoccum sp. cause stem-end rot of mango (Slippers et al., 2005). In Brazil, L. theobromae, F. aesculi, and N. parvum have been reported as pathogens of mango stem-end rot and dieback disease (de Oliveira Costa et al., 2010).
Fruits with typical symptoms were then selected for fun­gal isolation. The epidermis of fruit was first disinfested with 70% (v/v) ethanol and air-dried. Subsequently, small pieces (2-3 mm2) of necrotic tissue were dissected from the margins of lesions on fruit and placed on an acidified (750 μL of a 50% (v/v) solution of lactic acid per 300 mL of potato dextrose agar medium) (APDA) (Merck KGaA, Darmstadt, Germany). The plates were incubated at room temperature for 1-2 weeks. Putative Botryosphaeriaceae species isolates, recognized by their rapidly growing colo­nies with gray mycelium (Lazzizera et al., 2008), were subcultured on potato-dextrose agar (PDA) plates. Isolates were stored on PDA slants at 8°C. The frequency of oc­currence of the fungi in the collected fruits, which showed characteristic symptoms of fruit rot, was calculated ac­cording to the following formula: Frequency of occurrence (%) = (Number of fruits colonized by a specific pathogen/ Total number of fruits with fruit rot symptoms) x 100%.
Differentiation of Botryosphaeriaceae species were in the past chiefly based on the morphology of their ana-morphs (Jacobs and Rehner, 1998; Denman et al., 2000). However, morphological characteristics of these fungi may vary within the species, and in some cases, they may look very similar between species, making identi­fication of the fungus even more difficult. For efficient identification, DNA-based techniques have been applied to the taxonomy of Botryosphaeriaceae (Denman et al., 2000; Slippers et al., 2004; Alves et al., 2005; Taylor et al., 2005; Crous and Groenewald, 2005; Crous et al., 2006; De Wet et al., 2008). Combination of the molecular techniques with morphological characteristics has been used to successfully identify F. aesculi, N. parvum, and N. ribis, all of which were previously classified as F. aesculi (=B. dothidea) (sensu von Arx and Muller, 1954) (Jacobs and Rehner, 1998; Smith and Stanosz, 2001; Slippers et al., 2004; Crous et al., 2006).
Morphological characterization
For studies on colony morphology, isolates were grown on PDA and incubated at 25°C in darkness. The morphol­ogy of mycelium and conidia (dimensions, shape, color, presence of septa and longitudinal striations) were record­ed. To induce sporulation, putative Botryosphaeriaceae isolates were grown on 2% water agar plates containing sterilized pine (Pinus morrisonicola) needles (Smith et al., 1996), and incubated at 25°C with a 12-h light (near UV)/ dark cycle. For further purification, conidia released from pycnidia on the pine needles were spread on water agar. After 12-24 h, single germinating conidia were picked and transferred to PDA. Morphology of the conidia formed on pine needles was also examined under a stereoscopic mi­croscope (Nikon SMZ 1500, Tokyo, Japan). To determine the average length and width of conidia, at least 50 conidia from each isolate were analyzed using a light microscope (Nikon, ECLIPSE 80i, Tokyo, Japan); their images were photographed with a pixel camera system (Pixera Penguin 600CL, Los Gatos, CA, USA) and the length and width of conidia were measured by using a Simple PCI software Rev. 3.6 (Compix Inc., Cranberry Township, PA, USA). The measurements of conidia were subjected to statistical analyses and presented as average ± standard deviations.
Currently, fungal pathogens known to cause mango fruit rot include only Alternaria alternata, Phytophthora nicotianae, Pestalotiopsis mangiferae, and Phyllosticta anacardiacearum, according to "Common Names of Plant Diseases" posted on the website of the American Phyto-pathological Society (http://www.apsnet.org/publications/commonnames/Pages/
Mango.aspx). Information regarding the role of Botryosphaeriaceae species on mango fruit rot has been very limited. The aims of this study were to: a) identify Botryosphaeriaceae isolates collected from fruit rot of mango fruits, b) investigate the incidence of fruit rot and frequency of Botryosphaeriaceae species in three ma­jor mango producing areas of Taiwan, including Fanshan (Kaohsiung), Yujing, and Guntain (both in Tainan) during 2009-2011; c) test the pathogenicity and compare the viru­lence of Botryosphaeriaceae isolates obtained from mango fruits with fruit rot.
Molecular characterization
MATERIALS AND METHODS
The genomic DNA of fungal mycelia was isolated using the method described by Wang et al. (1993). For comparative phylogenetic study, partial sequences of three housekeeping genes were amplified by PCR, including ribosomal internal transcribed spacer (ITS), p-tubulin (TUB), and elongation factor 1-a (EF1-α). The PCR mixture contained 1X PCR buffer (10 mM Tns-HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% (w/v) gelatin, 1% Triton X-100), 100 μM of each dNTP, 0.2 μM of each primer, 0.4 U of Prozyme DNA polymerase (Protech Tech-nology Enterprise, Taipei, Taiwan). Primers used for the amplification of each gene were: ITS1 (5'-TCC GTA GGT
Field survey, disease symptoms, and fungal isolation
Field surveys were conducted at 77 orchards located in the Pintung (Fanshan) and Tainan (Yujing and Guntain) areas of Taiwan during 2009-2011, and 15-20 mango fruits were randomly collected from each orchard. The incidenc­es of fruit rot disease were calculated 7 days after harvest according to the following formula: Disease incidence (%) =(Number of fruits which showed only fruit rot but not anthracnose symptoms/ Total number of fruits) x 100%.
NI et al. ― New Botryosphaeriaceae fruit rot of mango
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GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') (White et al., 1990; Wang et al., 2010) for ITS, Bt2a (5'-GGT AAC CAA ATC GGT GCT GCT TTC-3') and Bt2b (5'-ACC CTC AGT GTA GTG ACC CTT GGC-3') for TUB (Glass and Donaldson, 1995), EF1-728F (5'-CAT CGA GAA GTT CGA GAA GG-3') and EF1-986R (5'-TAC TTG AAG GAA CCC TTA CC-3') for EF1-a (Carbone and Kohn, 1999), respectively. The amplification program included an initial step of 4 min at 94°C, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, and elongation at 72°C for 30 sec. A final extension was performed at 72°C for 10 min. The reaction was carried out by using a Bio-Rad iCycler Thermal cycler (Hercules, California, USA). The amplified products were sequenced in both directions by the dideoxy termination method through a service pro­vided by the Tri-I Biotech (Taipei, Taiwan).
developed 7 days after inoculation. Statistical analyses of the data were performed by using SAS (version 8, SAS Institute) with the Fisher's protected test, and an F value with P<0.05 was considered significant.
RESULTS
Field survey of mango fruit rot disease and collection of fungal isolates
To survey the occurrence of mango fruit rot disease, we collected mango fruits from three of the major mango producing areas in Southern Taiwan, including Fanshan (650 fruits from 32 orchards), Yujing (440 fruits from 22 orchards), and Guntain (454 fruits from 23 orchards). We examined all the fruits for the appearance of symptoms and found a total of 490 mango fruits with characteristic symptoms of fruit rot, which appeared as browning, soft, and watery lesions, different from that of anthracnose. The incidence of fruit rot disease identified in Guntain (46.0% in 2009, 53.8% in 2010, and 58.1% in 2011) was sigmfi-cantly greater than those found in Yujing (35.0% in 2009, 25.6% in 2010, and 23.8% in 2011) and Fanshan (20.1% in 2009, 18.7% in 2010, and 22.5% in 2011) (Table 1). The results indicated that this disease occurred frequently in all three mango producing areas.
For phylogenetic analysis, multiple sequence align­ment was performed with nucleic acid sequences of ITS rDNA, TUB, and EF1-α obtained from this study as well as those retrieved from GenBank (Table S1), by using the CLUSTAL X (Thompson et al., 1997), and a phylogram was generated by the application of neighbour-joining (NJ) algorithm in PAUP 4.0 beta 10 (Sinauer Associates, Sunderland, MA, USA). Bootstrap analysis was performed with 1000 replicates to test the branch strength.
Pathogenicity test
Isolates and morphological characterization
From the mango fruits showing symptoms of fruit rot (Figure 1), we obtained 237 isolates of Botryosphaeriaceae species. Based on their colony morphology on APDA plates, these Botryosphaeriaceae isolates were classified into four groups, and 5 to 10 isolates were randomly se­lected from each group for further studies.
Pathogenicity of Botryosphaeriaceae species isolated from diseased fruits was assessed by the use of either at­tached or harvested mango fruits. For inoculation of at­tached fruits in the orchard, 2 μL of conidial suspension (100 spores), which was prepared as described before, was dropped on the surface of an attached, immature mango fruit (cv. Irwin), followed by the addition of 20 μL water agar to immobilize the conidia. The inoculated fruit was then put inside a paper bag for protection. Each fungal iso­late was inoculated on twenty fruits. Four to 6 weeks post inoculation, the fruits were harvested and treated with cal­cium carbide to accelerate ripening. Incidence of mango fruit rot was recorded 2 weeks after harvest.
When cultured on PDA or water agar containing pine needles, most isolates sporulated within 21 days of incu­bation. No teleomorph was observed for any isolate dur­ing this study. Based on the morphology of colonies and conidia, all Botryosphaeriaceae isolates were classified as one of four distinct species, including L. theobromae, N. mangiferae, N. parvum and F. aesculi (Table 2).
For inoculation of the harvested fruits, a total of nine isolates representing four different species of Botry-osphaeriaceae were selected for pathogenicity tests. For inoculation, fungal isolates were grown on PDA at 25°C for 7 days. Unripe but mature mango fruits (cv. Irwin) were collected from Yujing (Tainan, Taiwan) and treated with hot water (60°C) for 20 sec to avoid possible inter­ference from latent infections of postharvest pathogens. Prior to inoculation, fruits were pricked with a sterile needle, and small discs of agar (5 mm in diameter) colo­nized by Botryosphaeriaceae isolates were placed on the wound. Each fungal isolate was inoculated on three fruits. As a control, a parallel experiment was performed by covering the wounded zone with discs of PDA without Botryosphaeriaceae. Pathogenicity of the fungal isolates was determined based on the length of lesions (mm) that
When subcultured on PDA, L. theobromae initially produced white, fluffy aerial mycelium that rapidly cov­ered the surface of Petri dishes within two days of in­cubation. The mycelium then turned to pale olivaceous gray within 3-4 days, and produced pycnidia after 7 days. When visualized from the bottom of the Petri dish, the colonies first showed white to olivaceous gray, and became dark olivaceous after 7-10 days. It produced py-cnidia while incubated in water agar (WA) supplemented with sterilized pine needles after 7-14 days. Immature conidia were hyaline, aseptate, with the shape of ellipsoid to ovoid. They became uniseptate, thick-walled, light brown pigmented with longitudinal striations once mature (Figure 1A). The average length (L) and width (W) of 400 conidia was 23.40-27.18 x 12.47-15.08 μm, with an L/W ratio of 1.61-1.98 (Table S2).
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Table 1.
The incidence of fruit rot disease and occurrence frequency of Botryosphaeriaceae species on mango fruits exhibiting
symptoms of fruit rot during 2009-2011.
Region
Year
Incidence (%)a
Frequency of occurrence (%)b
Lasiodiplodia theobromae
Neofusicoccum parvum
Neofusicoccum mangiferae
Fusicoccum aesculi
Fanshan
2009
20.1
1.8
16.1
28.6
12.5
2010
18.7
6.4
25.5
4.3
34.0
2011
22.5
0.0
37.8
20.0
37.8
Yujing
2009
35.0
9.7
3.2
3.2
25.8
2010
25.6
2.8
30.6
0.0
47.2
2011
23.8
10.5
26.3
7.9
13.2
Guntain
2009
46.0
7.5
0.0
2.5
17.5
2010
53.8
3.3
5.0
0.0
8.3
2011
58.1
13.1
3.6
2.2
12.4
aIncidence of mango fruit rot in each area was calculated by the following formula: Disease incidence (%) = (Number of fruits which showed only symptoms of fruit rot but not anthracnose/Total number of fruits) x 100%.
^Frequency of occurrence (%) = (Number of fruits colonized by a pathogen/Total number of fruits with fruit rot symptoms) x 100%.
Figure 1.
Symptoms of mango fruit rot and morphology of fungal colony, pycnidia, and conidia. A: Lasiodiplodia theobromae; B:
Neofusicoccum mangiferae; C: Fusicoccum aesculi; D: N. parvum. Bar = 10 μm.
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Table 2.
Representative isolates of Botryosphaeriaceae species collected from mango fruits with fruit rot in southern Taiwan.
GenBank Accession No.
Isolate
Identity
Locality
ITS
EF1-α
TUB
B961
Lasiodiplodia theobromae
Guntain
GQ502453
GQ979999
GU056845
B965
L. theobromae
Guntain
GQ502454
GQ980000
GU056854
B838
L. theobromae
Fangshan
GQ502456
GQ980001
GU056852
B852
L. theobromae
Guntain
GQ502457
GQ980002
GU056851
B918
L. theobromae
Guntain
GQ502458
GQ980003
GU056850
B902
L. theobromae
Guntain
GQ502459
GQ980004
GU056849
B878
L. theobromae
Guntain
GQ502460
GU056848
B607
L. theobromae
Guntain
GQ502461
GU056846
B845
Neofusicoccum parvum
Fangshan
GQ861434
GQ985316
GU062771
B946
N. parvum
Yujing
GQ861432
GQ985313
GU062768
B1314
N. parvum
Fangshan
GU073291
GU121436
GU111537
B794
N. parvum
Fangshan
GQ861433
GQ985312
GU062767
B1260
N. parvum
Fangshan
GU073287
GU121432
GU111533
B1272
N. parvum
Yujing
GU073288
GU121433
GU111534
B1296
N. parvum
Yujing
GU073289
GU121434
GU111535
B1307
N. parvum
Fangshan
GU073290
GU121435
GU111536
B809
N. mangiferae
Fangshan
GQ848323
GQ998898
GU071122
B793
N. mangiferae
Fangshan
GQ848320
GQ998900
GU071120
B808
N. mangiferae
Fangshan
GQ848322
GQ998899
GU071121
B979
N. mangiferae
Yujing
GQ848315
GQ998897
GU071123
B763
N. mangiferae
Fangshan
GQ421486
GQ998896
GU071119
B964
Fusicoccum aesculi
Guntain
GQ861429
GU002157
GU071124
B811
F. aesculi
Fangshan
GU453689
GU002164
GU071125
B844
F. aesculi
Fangshan
GU453690
GU002163
GU071126
B922
F. aesculi
Yujing
GQ421485
GU002162
GU071127
B833
F. aesculi
Fangshan
GQ861430
GU002161
GU071129
B801
F. aesculi
Fangshan
GQ861431
GU002160
GU071130
B932
F. aesculi
Yujing
GQ861428
GU002159
GU071131
B1113
F. aesculi
Yujing
GU453691
GU002158
GU071132
Isolates of N. mangiferae initially produced white, ap-pressed mycelium. Four days after incubation on PDA, the middle of colony turned to pale olivaceous gray and produced pycnidia 10-14 days after incubation. When vi­sualized from the bottom of Petri dish, the colonies were olivaceous to black. This fungus grew slower than the other three Botryosphaeria species found in this study. Pycnidia were formed on WA supplemented with sterilized pine needles after 3-7 days. The conidia were hyaline and ovoid (Figure 1B), with an average length and width of 11.98-12.93 x 6.25-6.98 μm (L/W= 1.85-1.95) (Table S2).
days. Pycnidia were formed on WA supplemented with sterilized pine needles after 1-2 weeks. Conidia were hya­line, thin-walled, aseptate, and fusiform (Figure 1C), with an average length and width of 18.72-22.10 x 5.72-6.63 £gm (L/W= 3.05-3.52) (Table S2).
Cultures of N. parvum were initially white with aerial mycelium. The middle of colony then became pale oliva­ceous gray after 3-4 days. When visualized from the bot­tom of Petri dish, the colony was deep olivaceous gray to black after 4-7 days of incubation. Pycnidia were formed on WA supplemented with sterilized pine needles for 2-3 weeks. Conidia were hyaline, aseptate, and fusiform (Fig­ure 1D). The average length and width of 416 conidia were 15.85-19.25 x 4.49-6.61 μm (L/W= 2.70-3.68) (Table S2).
Colonies of F. aesculi were initially white with aerial mycelium. They became pale olivaceous gray from the center of colony after 3-4 days, and turned black after 7
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Phylogenetic analysis
nucleotide sequence of ITS, TUB, and EF1-α showed a respective identity range of 93-98%, 91-98%, and 75-96% among different species.
To confirm the morphometric identifications and to infer the evolutionary relatedness among these Botry-osphaeriaceae species, partial nucleotide sequences of ITS, TUB, and EF1-a from L. theobromae, N. mangiferae, N. parvum, and F. aesculi were amplified by PCR. The respective length of amplified products of ITS, TUB, and EF1-a for L. theobromae were approximately 540, 460, and 320 bp, respectively, while those for N. mangiferae, N. parvum, and F. aesculi were 580, 460, and 300 bp, respectively. Sequences of these amplified products were compared with those deposited in GenBank (Table 2), and performed multiple sequence alignments to detect differ­ences among these species. Sequences of the ITS, TUB, and EF1-a genes from isolates of the same species showed an identity ranging from 98% to 1 00%. In contrast,
In addition, three unrooted phylogenetic trees were constructed based on multiple sequence alignment of ITS, TUB, and EF1-a. The overall topology of these trees was similar, with each tree composed of four major clades (Figure 2). Isolates from the same species formed a single, monophyletic group with a bootstrap support ranging from 90% to 100%. Moreover, the clade representing N. mangiferae is close to that of N. parvum in all three trees.
Frequency of Botryosphaeriaceae species in southern Taiwan
To know the frequency of occurrence of each fungal pathogen in different mango producing areas, the per-
Figure 2.
Unrooted phylogenetic trees of Botryosphaeriaceae
species based on the nucleotide sequences of internal transcribed spacer (ITS) (A), p-tubulin (TUB) (B), and elongation factor 1-a (EF1-α) (C). Sequence alignments were conducted by using Clustal X, and phylogenetic trees were then constructed by the neighbor-joining method. Bootstrap values from 1000 replicates were given above the nodes. Lt: Lasiodiplodia theobromae; Fa: Fusicoccum aesculi; Np: Neofusicoccum parvum; Nm: Neofusicoccum mangiferae.
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centage of fungal isolates collected from the diseased mango fruits were calculated. As shown in Table 1, at least three Botryosphaeriaceae species were found in Fan-shan, Yujing, and Guntain, respectively, but their relative prevalence was different. In 2009, the major pathogen in Fanshan was N. mangiferae, while that for Yujing and Guntain was F. aesculi. In 2010, F. aesculi and N. parvum were predominant in Fanshan and Yujing. They were also found in Guntain, but only with a low frequency. In 2011, both F. aesculi and N. parvum were found frequently in Fanshan, while only N. parvum was predominant in Yu-jing. In Guntain, both L. theobromae and F. aesculi oc­curred more frequently than N. mangiferae and N. parvum. It was obvious that, even in the same region, incidence of mango fruit rot caused by some pathogens differed greatly between years. For example, the occurrence frequency of N. mangiferae in Fanshan varied from 28.6% in 2009 to 4.3% in 2010, and then to 20.0% in 2011. Moreover, the frequency of N. mangiferae in Yujing and Guntain during 2009-2011 was less than 8% and 3% of the total rotted mangos, respectively.
Table 4.
Mean length of lesions caused by the isolates of
Botryosphaeriaceae species following inoculation on wounded or unwounded mango fruits (cultivars 'Irwin') for 7 days.
Mean lesion
Mean lesion
Isolate
length (mm)a
length (mm)
(wounded)
(unwounded)
Neofusicoccum mangiferae (B763)
43.4 bc
18.9 b
Neofusicoccum mangiferae (B793)
42.3 bc
9.7 b
Fusicoccum aesculi (B811)
41.4 c
11.3 b
Fusicoccum aesculi (B932)
45.5 bc
8.0 b
Lasiodiplodia theobromae (B826)
101.9 a
56.7 a
Lasiodiplodia theobromae (B878)
114.4 a
73.3 a
Neofusicoccum parvum (B837)
66.5 b
55.3 a
Neofusicoccum parvum (B1001)
60.8 bc
26.0 b
Neofusicoccum parvum (B010)
60.4 bc
29.3 b
LSD (P = 0.05)
25.0
25.4
a Means followed by the same letter are not significantly different.
Pathogenicity tests
To evaluate the pathogenicity of the Botryosphaeri-aceae isolates, inoculation experiments on attached, immature mango fruits was performed with conidial suspensions of L. theobromae, N. mangiferae, N. parvum or F. aesculi. Four to 6 weeks after inoculation, the in­oculated fruits were harvested and ripened by a treatment using calcium carbide. Fruit rot symptoms appeared on the fruit body as fruits ripened gradually, no matter which pathogen was used as the inoculum. When examined 2 weeks after harvest, most of the inoculated fruits showed symptoms characteristic of fruit rot (Table 3). In contrast, the mock-inoculated fruits appeared healthy and intact. To make sure that lesions on the diseased fruits were caused by Botryosphaeriaceae species, the pathogens were reiso-lated from the lesion edge and examined as described in
the previous section. These results indicated that symp­toms development on inoculated fruit was indeed caused by the specific Botryosphaeriaceae isolate originally used as the inoculum.
Furthermore, inoculation experiments were also per­formed with harvested mango fruits. When inoculated on the harvested fruits, all Botryosphaeriaceae isolates re­sulted in the formation of black-brown lesions of irregular shape on the surface of both wounded and unwounded fruits within 7 days post inoculation. In contrast, no le­sion developed on wounded or unwounded fruits of the control experiment. Recovery of fungal isolates from the lesion edge of the diseased fruits confirmed that the fruit rot symptoms was indeed caused by a Botryosphaeriaceae isolate which was used as inoculum. Measurement of le­sion lengths on the inoculated mango fruits followed by statistical analyses indicated that the size of lesions caused by F. aesculi, N. mangiferae, and N. parvum showed no significant difference on both wounded and unwounded fruits (Table 4). In contrast, the lesion size caused by L. theobromae was significantly larger than those of the other three pathogens. Nonetheless, when the inoculation experi­ment was performed on unwounded fruits, isolates of the same Botryosphaeriaceae species differed in virulence as seen in the case of N. parvum. The size of lesions caused by isolate B837 on unwounded fruits (55.3 mm) was sig­nificantly larger than those caused by the other two N. par­vum isolates, B1001 and B010, but showed no significant difference from those of L. theobromae. It is also worth mentioning that, when the inoculation was performed with wounded fruits, the lesions caused by all four pathogens developed faster and ended up with the formation of le­sions with a bigger size compared to those observed on unwounded fruits (Table 4).
Table 3.
Incidence of fruit rot on mature mango fruits after
inoculation of attached mango fruits with L. theobromae, N. mangiferae, F. aesculi, or N. parvum.
Pathogen
No. diseased/ No. Exp. 1
harvested fruitsb Exp. 2
L. theobromae
18/20
17/20
N. mangiferae
20/20
12/15
F. aesculi
15/17
15/19
N. parvum
17/20
18/20
Control
0/20
0/15
aConidia of the fungal pathogen were inoculated on the imma­ture fruits which were still attached on the mango trees.
bFour to 6 weeks post inoculation, the fruits were harvested and ripened by the treatment with calcium carbide. Incidence of fruit rot was recorded 2 weeks after harvest of the mango fruits.
474
Botanical Studies, Vol. 53, 2012
DISCUSSION
survey. N. parvum is known to be associated with stem-end rot of mango in Australia (Slippers et al., 2005) and Brazil (de Oliveira-Costa et al., 2010), and dieback of mango in Peru (Javier-Alva et al., 2009). In addition, it has been reported as the pathogen of stem canker and dieback of Asian pear trees in Taiwan (Shen et al., 2010). On the other hand, F. aesculi is known to cause canker and fruit rot in numerous woody plants, such as fruit rot of olives (Phillips et al., 2005), canker in grapevines (Urbez-Torres et al., 2006), shoot and panicle blight in eucalyptus (Yu et al., 2009), stem-end rot of mango in Brazil (de Oliveira Costa et al., 2010) and Australia (Slippers et al., 2005). In Taiwan, F. aesculi caused the ring rot of Pyrus and stem canker of Salix (Tsai et al., 1991). In this study, we found that, in 2010 and 2011, N. parvum and F. aesculi were the major Botryosphaeriaceae species that caused mango fruit rot in Fanshan and Yujing. In 2009, the dominant pathogen in Fanshan was N. mangiferae, while that in Yujing was F. aesculi. N. mangiferae has been found as the pathogen of stem-end rot of mango (Slippers et al., 2005), and the ca­sual agent of avocado fruit rot in Taiwan (Ni et al., 2009). The reason determining which fungus is the dominant pathogen for causing mango fruit rot can be complicated, because factors such as survival of fungus in the field, inoculum source, inoculum density, climate, and cultiva­tion practices all could affect the dynamics of pathogens as well as the frequency of disease incidence in the field (Niekerk et al., 2011; Sakalidis et al., 2011).
Mango fruit rot is characterized by the appearance of brown to dark spots on the epidermis of the fruit body. Subsequently, the affected areas will rapidly split open and become soft and watery. Therefore, once infected, mango fruits completely lose their commercial values. As revealed by its high incidence, fruit rot has become an important post-harvest disease of mango in Taiwan. However, little is known about pathogens causing this disease. Based essen­tially on characteristics of the anamorph, such as morphol­ogy of fungal colonies and conidia, we have identified four pathogens which belong to Botryosphaeriaceae, including L. theobromae, N. mangiferae, N. parvum, and F. aesculi. Nonetheless, F. aesculi and N. parvum remained difficult to identify, because of their similarity in colony morphol­ogy as well as size and shape of conidia. Indeed, these two species have in former years been treated as part of the B. dothidea complex (Smith and Stanosz, 2001; Crous et al., 2006). In this study, phylogenetic analysis of the sequenc­es of ITS, TUB, and EF1-a clearly separated N. parvum from F. aesculi, indicating that molecular characteristics were useful for the differentiation of Botryosphaeriaceae species. In support of our results, L. theobromae, F. aes-culi, N. mangiferae, and N. parvum are known to form dis­tinct clades by phylogenetic analysis (Slippers et al., 2005; Damm et al., 2007; de Oliverira Costa et al., 2010).
In culture, isolates of L. theobromae grew much faster than the other three fungal species, able to fully colonize a 90-mm Petri dish within 48 h. Furthermore, they produced conidia with pigment and longitudinal striations, which were quite different from those of the other species. As an important pathogen of woody hosts, L. theobromae has been reported to cause cankers, dieback, fruit rot, and root rots on over 500 different hosts, including perennial fruits, nut trees, vegetable crops, and ornamental plants (Punithal-ingam, 1980; Alves et al., 2008; Urbez-Torres et al., 2008; Abdollahzadeh et al., 2010). In Taiwan, L. theobromae is also known to cause stem canker and fruit rot of guava as well as stem-end rot of mango and papaya (Wang and Hsieh, 2006; Wang et al., 2007). In the present study, we verified that L. theobromae is one of the fungal pathogens that causes mango fruit rot. Moreover, as revealed by the inoculation experiment, L. theobromae was more virulent than the other three Botryosphaeriaceae pathogens, which was also noticed by de Oliverira-Costa et al. (2010). It is also interesting to notice that, despite L. theobromae being highly virulent, incidence of fruit rot caused by this patho­gen was relatively low in Fanshan, Yujing, and Guntain, compared to those caused by the other three pathogens. It is likely that the inoculum density of L. theobromae was low in these areas. Further investigation will be conducted to investigate the relationship between the inoculum den­sity and disease incidence.
Furthermore, it is also interesting to note that the to­tal frequency of Botryosphaeriaceae species in Guntain was lower than that in Fanshan and Yujing, despite of the high incidence of fruit rot in this area. Indeed, Phomopsis spp. were isolated at a high rate in Guntain, suggesting the roles of additional fungal pathogens other than Botry-osphaeriaceae species as causal agents of mango fruit rot (unpublished data).
Although the causal agents of both mango fruit rot and stem-end rot are L. theobromae, F. aesculi, N. mangiferae, and N. parvum, fruit rot is a disease distinct from stem-end rot. Johnson and Cooke (1991) suggested that these pathogens may occur as endophytes in mango stem tissue and colonize the stem end of mango fruits during matura­tion, thereby causing stem-end rot. In this study, however, lesions caused by Botryosphaeriaceae species were usually found on the fruit body, suggestive of an infection pathway different from that of stem-end rot. Inoculum may come from conidial ooze generated from dead twigs of mango trees (unpublished data), or fungal spores in soil and leaf litter around mango orchards (Johnson, 2008). In sup­port of this idea, as shown in the inoculation experiments performed with attached, immature mango fruits, conidia of these Botryosphaeriaceae species were able to infect the fruits successfully. Therefore, to reduce the incidence of fruit rot, it is important to remove the dead twigs and branches for routine maintenance of orchard hygiene, and also to wrap developing fruits inside paper bags for pro­tection against conidia residing in soil and diseased plant
Neofusicoccum parvum and F. aesculi, both are first reported as pathogens of mango in Taiwan, were the domi­nant species associated with mango fruit rot during this
NI et al. ― New Botryosphaeriaceae fruit rot of mango
475
tissues on the ground. When the inoculation experiments were performed with wounded mango fruits, the symp­toms developed faster than that on unwounded fruits. It is likely that fruit sap released by the wounds may serve as nutrients of the mycelium, and thus lead to rapid growth of the fungus onto the plant tissue (Amponsah et al., 2011). It is thus also important to avoid the making of any wound on the mango fruits to reduce the incidence of fruit rot.
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The present work is the first comprehensive study of Botryosphaeriaceae species that are associated with mango fruit rot. Correct identification of these pathogens is helpful for more effective management of fruit rot dis­ease of mango. Further screening of effective fungicides and understanding of the epidemiology of these fungal pathogens will help to reduce financial loss to the mango industry in Taiwan.
Acknowledgement.
This study was supported by the
Council of Agriculture of Taiwan. We thank S. L. Hsu and S. Y. Lai for their excellent technical supports.
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NI et al. ― New Botryosphaeriaceae fruit rot of mango
477
Botryosphaeriaceae¦b»OÆW¤Þ°_¤§ÂcªGªG»G¯f¡G¯f­ìºØÃþ
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°Ï¡C¦ÛªG»G¯f´³©Ò¤ÀÂ÷¤§¯f­ìµß¸g¥H§ÎºA¯S©Ê¤ÎITS, β-tubulin (TUB)»PEF1-αµ¥°ò¦]¤§§Ç¦CŲ©w«á¡A¦@
µo²{¥|ºØ¸²µå®yµÄµß¬ì¯f­ìµß¡A¥]¬ALasiodiplodia theobromae, Neofusicoccum mangiferae, N. parvum¤Î Fusicoccum aesculi ¡C¤£½×¬Oª½±µ±µºØ©Î¥ý»s³y¶Ë¤f¦A±µºØ¡A³o¥|ºØ¯f­ìµß³£¥i¦¨¥\·P¬VÂcªGªG¹ê¡AÅã¥Ü
¨ä§¡¨ã¦³¯f­ì©Ê¡A¦Ó¥BL. theobromae¤§­P¯f¤O¸û¨ä¥¦¤TºØ¯f­ìµßÁÙ±j¡C·í¥H¥Íªø©óÂcªG¾ð¤§¥¼¦¨¼ôªG
¹ê¶i¦æ±µºØ¸ÕÅç®É¡Aµo²{¤£»Ý»s³y¶Ë¤f ¡A¯f­ìµß¤§¤À¥Í­M¤l§Y¥iª½±µ·P¬VªG¹ê¡A¦Ó¥B±Ä¦¬¤§ªG¹ê¦b¦¨¼ô
«á¤]·|¥X²{ªG»G¯f´³¡C¥»¬ã¨sÃÒ©úÂcªGªG»G¯f¤D¥ÑL. theobromae, F. aesculi, N. mangiferae¤ÎN. parvum
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Table S1. Sequences of Botryosphaeriaceae species from GenBank used in the phylogenetic analysis.
Species
GenBank Accession No.
ITS
EF1-α
TUB
Lasiodiplodia theobromae
AY640255
AY236901
AY236930
DQ458891
AY640258
EU673110
GQ502452
GQ980005
GU056847
GQ502455
GU056853
Neofusicoccum parvum
AY259098
AY573221
AY615169
AY615182
DQ487158
EU673095
GQ861435
GQ985315
GU062770
N. mangiferae
AY615186
DQ093221
AY615172
DQ316081
AY615173
Fusicoccum aesculi
AY615191
EF585562
AY615178
(Botryosphaeria dothidea)
EF638769
EF638727
EU673106
478
Botanical Studies, Vol. 53, 2012
Table S2.
Dimensions of conidia of selected isolates of Botryosphaeriaceae species collected from mango fruits with fruit rot.
Isolate
Identity
Conidium length (μm± SD)
Conidium width (μm± SD)
L/W
B961
Lasiodiplodia theobromae
24.35±1.48(50)a
12.47±1.40
1.98±0.32
B965
L. theobromae
23.40±1.62(50)
12.75±0.95
1.84±0.18
B838
L. theobromae
24.86±1.38(50)
14.42±0.78
1.73±0.12
B852
L. theobromae
24.99+1.90(50)
15.59+0.87
1.61 +0.15
B918
L. theobromae
23.49±1.22(50)
12.90±0.66
1.82±0.13
B902
L. theobromae
24.73±1.98(50)
13.35±0.99
1.86±0.18
B878
L. theobromae
24.77+1.19(50)
13.07+0.77
1.87+0.16
B607
L. theobromae
27.18+1.86(50)
15.08+1.75
1.82+0.21
B845
Neofusicoccum parvum
15.85±1.16(58)
4.49±0.11
3.52±0.30
B946
N. parvum
16.54±1.63(50)
5.57±0.40
2.97±0.28
B1314
N. parvum
17.71±1.68(56)
6.61±0.80
2.70±2.24
B794
N. parvum
18.87±2.11(50)
5.25±0.65
3.65±0.65
B1260
N. parvum
19.25±1.53(50)
5.97±0.54
3.26±0.37
B1272
N. parvum
19.24±2.23(50)
6.13±1.52
3.26±0.40
B1296
N. parvum
18.50±1.65(52)
5.07±0.57
3.68±0.52
B1307
N. parvum
19.08±1.34(50)
5.68±0.48
3.39±0.47
B809
N. mangiferae
12.01±0.94(50)
6.35±0.55
1.86±0.20
B793
N. mangiferae
11.98±0.80(50)
6.25±0.47
1.92±0.15
B808
N. mangiferae
12.26±0.77(60)
6.37±0.34
1.93±0.13
B979
N. mangiferae
12.50±1.00(50)
6.42±0.48
1.95±0.15
B763
N. mangiferae
12.93±0.93(50)
6.98±0.40
1.85±0.13
B964
Fusicoccum aesculi
21.56±2.18(50)
6.40±2.22
3.50±0.53
B811
F. aesculi
19.31±1.59(50)
6.10±0.36
3.17±0.26
B844
F. aesculi
19.86±1.20(50)
6.38±0.36
3.12±0.24
B922
F. aesculi
22.10±1.92(50)
6.28±0.30
3.52±0.30
B833
F. aesculi
18.72±2.70(50)
6.14±0.36
3.05±0.43
B801
F. aesculi
19.67±1.04(50)
5.72±0.38
3.46±0.31
B932
F. aesculi
21.34±1.47(50)
6.63±0.70
3.26±0.41
B1113
F. aesculi
20.51±1.31(54)
5.92±0.32
3.48±0.31
aNumerals inside the parenthesis indicate the number of conidia used for the measurement of length and width.