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Keywords:

  • Fusarium solani;
  • IGS;
  • ITS;
  • orchids;
  • pathogenicity;
  • β-tubulin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The pathogenicity of 35 Fusarium solani isolates obtained from diseased leaves of greenhouse-grown Phalaenopsis plants in Taiwan was tested on different orchids, including Phalaenopsis sp., Cymbidium spp., Oncidium sp., Dendrobium sp. and Cattleya sp., plus pea (Pisum sativum), chrysanthemum (Chrysanthemum indicum) and cucurbit [melon (Cucumis melo) and cucmber (C. sativus)] plants. Isolates of F. solani from Phalaenopsis spp. caused severe leaf yellowing on Phalaenopsis and mild symptoms on Cymbidium spp., but no visual symptoms on Oncidium sp., Dendrobium sp., Cattleya sp., pea, chrysanthemum or melon. Fusarium solani isolates collected from Phalaenopsis, pea and cucurbits were molecularly characterized by internal transcribed spacer (ITS), intergenic spacer (IGS) and β-tubulin gene analyses. Phylogenetic trees constructed by distance and parsimony methods indicated that isolates from Phalaenopsis were grouped into one type based on ITS, IGS and β-tubulin sequences with high bootstrap value (> 84%) support, compared to ‘formae speciales’ of F. solani from the other hosts. These analyses show that isolates of F. solani from Phalaenopsis are distinct from F. solani isolates from other hosts in Taiwan. Therefore, it is proposed that F. solani isolates that incite Phalaenopsis leaf yellowing be designated F. solani f. sp. phalaenopsis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Phalaenopsis spp. constitute a major component of the export flower market in Taiwan (Yoneda, 2007). Several fungal diseases of Phalaenopsis have been reported in Taiwan, including diseases caused by Fusarium solani, F. oxysporum and F. proliferatum (Hsu et al., 2002; Chen & Chung, 2008; Chen et al., 2008, 2009). Among the Fusarium pathogens, F. solani, which causes leaf yellowing, basal rot and root rot, is the predominant pathogen of greenhouse-grown Phalaenopsis in Taiwan (Chen & Chung, 2008; Chen et al., 2008, 2009).

Fusarium solani is an important soilborne pathogen with worldwide distribution (Joffe & Palti, 1977; Samac & Leong, 1989; Jeschke et al., 1990; Leslie et al., 1990; Ploetz, 1991; Pegg et al., 2002; Chen et al., 2006). Based on host specificity, Suga et al. (2000) divided F. solani into 10 ‘formae speciales’ (f. sp.) including batatas, cucubitae, eumartii, mori, phaseoli, piperis, pisi, radicicola, robiniae and xanthoxyli. Previous studies on pathogenicity of Fsolani from orchids have focused on Phalaenopsis (Morita et al., 1992; Kim et al., 2002; Lin et al., 2007) without reference to other orchids. Thus, little is known about host specificity of F. solani from Phalaenopsis among orchids.

Knowledge about genetic diversity and phylogeny among Fusarium spp. is based on DNA-based techniques including internal transcribed spacer (ITS) (Suga et al., 2000), amplified fragment length polymorphisms (AFLPs) (Baayen et al., 2000; Belabid et al., 2004; Leslie et al., 2004) and elongation factor-1 alpha (EF-1α) (O’Donnell et al., 1998) analyses. These techniques have proven useful to establish phylogenetic relationships among Fusarium spp. and ‘formae speciales’. The objectives of this study were: (i) to determine whether F. solani isolates from Phalaenopsis are pathogenic to other orchids, pea, cucumber, melon and chrysanthemum, and (ii) to assess molecular phylogenetic relationships among F. solani isolates from Phalaenopsis and those from other hosts by making rDNA-ITS, rDNA-IGS and β-tubulin sequence comparisons.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal isolates

Forty-nine F. solani isolates were evaluated in this study, including 35 isolates from Phalaenopsis amabilis and Phalaenopsis spp. leaves, five isolates from pea roots (Pisum sativum), four isolates from melon roots (Cucumis melo), two isolates from Catharanthus roseus and three isolates from chrysanthemum roots (Chrysanthemum indicum) (Table 1). Stock cultures of isolates used in this study were derived from single microconidia from original isolates and maintained at 4°C on potato dextrose agar (PDA).

Table 1.   Host and location of 49 isolates of Fusarium solani collected in Taiwan
HostIsolateLocationDate of collectionAccession number in DDBJ/NCBI/EMBL GenBank
ITSIGSβ-tubulin
  1. NT: Not tested.

Phalaenopsis amabilis cv. W-10YS11bDalin, Chayi04/05/2007AB551689AB551818AB553608
P. amabilis cv. W-10YS12bDalin, Chayi04/05/2007AB551690AB551819AB553609
P. amabilis cv. W-10YS13aDalin, Chayi04/05/2007AB551691AB551820AB553601
P. amabilis cv. W-10YS21aDalin, Chayi04/05/2007AB551692AB551821AB553611
P. amabilis cv. W-10YS21bDalin, Chayi04/05/2007AB551693AB551822AB553612
P. amabilis cv. W-10YS22aDalin, Chayi04/05/2007AB551694AB551823AB553613
P. amabilis cv. W-10YS31aDalin, Chayi04/05/2007AB551695AB551824AB553614
Phalaenopsis sp. cv. Sogo YukidianYS31bDalin, Chayi04/05/2007AB551696AB551825AB553615
Phalaenopsis sp. cv. Sogo YukidianYPE2cDalin, Chayi09/01/2008AB551685AB551814AB553604
Phalaenopsis sp. cv. Sogo YukidianYPE4dDalin, Chayi09/01/2008AB551686AB551815AB553605
Phalaenopsis sp. cv. Sogo YukidianYPE5aDalin, Chayi09/01/2008AB551687AB551816AB553606
Phalaenopsis sp. cv. Sogo YukidianYPE6bDalin, Chayi09/01/2008AB551688AB551817AB553607
Phalaenopsis sp. cv. Sogo WeddingSJ2aMeinung, Kaohsiung29/08/2007AB551682AB551810AB553600
Phalaenopsis sp. cv. Sogo WeddingSJ2bMeinung, Kaohsiung29/08/2007AB551683AB551811AB553601
P. amabilis cv. Han-BenCPY-01aWanluan, Pingtung28/08/2007AB551678AB551806AB553596
P. amabilis cv. Han-BenCPY-01cWanluan, Pingtung28/08/2007AB551679AB551807AB553597
P. amabilis cv. Han-BenSLuY-01aSinpi, Pingtung28/08/2007AB551680AB551808AB553598
P. amabilis cv. Han-BenSLuY-01cSinpi, Pingtung28/08/2007AB551681AB551809AB553599
Phalaenopsis sp. cv. Sogo WeddingFM2bHoubi, Tainan22/06/2007AB551665AB551793AB553583
Phalaenopsis sp. cv. Sogo YukidianFN2aYanshuei, Tainan22/06/2007AB551666AB551794AB553584
Phalaenopsis sp. cv. Sogo YukidianFN2bYanshuei, Tainan22/06/2007AB551667AB551795AB553585
Phalaenopsis sp. cv. Sogo YukidianFN2cYanshuei, Tainan22/06/2007AB551668AB551796AB553586
Phalaenopsis sp. cv. Sogo YukidianSM1aMadou, Tainan19/07/2007AB551702AB551812AB553603
Phalaenopsis sp. cv. Sogo YukidianSM1bMadou, Tainan19/07/2007AB551684AB551813AB553602
Phalaenopsis sp. cv. Sogo WeddingCYY3aHoubi, Tainan02/07/2007AB551697AB551826AB553616
Phalaenopsis sp. cv. Sogo WeddingCYY3cHoubi, Tainan02/07/2007AB551698AB551827AB553617
Phalaenopsis sp. cv. Sogo YukidianFS1aTapi, Yulin04/05/2007AB551669AB551797AB553587
Phalaenopsis sp. cv. Sogo YukidianFS1bTapi, Yulin04/05/2007AB551670AB551798AB553588
Phalaenopsis sp. cv. Sogo YukidianFS3aTapi, Yulin04/05/2007AB551671AB551799AB553589
Phalaenopsis sp. cv. Sogo YukidianFS3bTapi, Yulin04/05/2007AB551672AB551800AB553590
Phalaenopsis sp. cv. Sogo YukidianJJ1aTapi, Yulin04/05/2007AB551673AB551801AB553591
Phalaenopsis sp. cv. Sogo YukidianJJ2aTapi, Yulin04/05/2007AB551674AB551802AB553592
Phalaenopsis sp. cv. Sogo YukidianJJ2bTapi, Yulin04/05/2007AB551675AB551803AB553593
Phalaenopsis sp. cv. Sogo YukidianJJ3aTapi, Yulin04/05/2007AB551676AB551804AB553594
Phalaenopsis sp. cv. Sogo YukidianJJ3bTapi, Yulin04/05/2007AB551677AB551805AB553595
Pisum sativumFS-3Fushing, Changhua14/01/1997NTAB551833NT
FS-4Fushing, Changhua14/01/1997AB551705NTAB553623
FS-5Fushing, Changhua14/01/1997AB551706NTAB553624
FS-6Fushing, Changhua14/01/1997AB551707NTAB553625
FS-8Erlin, Changhua26/09/1997NTAB551834NT
Catharanthus roseusFS01pTaichung2005AB551703AB551831AB553621
FS02pTaichung2005AB551704AB551832AB553622
Chrysanthemum indicumFS-53Tianwei, Changhua18/10/1998AB551708AB551835AB553626
FS-54Tianwei, Changhua18/10/1998AB551709AB551836AB553627
FS-55Tianwei, Changhua18/10/1998AB551710NTAB553628
Cucumis meloFS-57Tungsan, Tainan06/04/1999AB551711AB551837AB553629
FS-61Wufeng, Taichung08/11/1999AB551712AB551838AB553630
FS-64Tainan04/12/1999AB551713AB551839AB553630
FS-65Tainan04/12/1999AB551714AB551840AB553632

Test for pathogenicity of F. solani isolates from Phalaenopsis to other orchids

Pathogenicity of 10 Phalaenopsis isolates of F. solani was confirmed on Phalaenopsis cv. V3 (Phalaenopsis sp. cv. Sogo Yukidian), the most available cultivar in Taiwan, and then evaluated for pathogenicity to other orchids, including Cattleya sp., Cymbidium spp., Dendrobium sp. and Oncidium sp. Prior to inoculation, plants were propagated on fern chips in pots (15 cm diameter; one plant per pot) in the greenhouse at 28°C for 1 month. Inoculation involved making three puncture wounds at the leaf base with a sterile needle followed by pipetting 100 μL conidial suspension (105 spores mL−1) in 0·1% Tween 20) prepared from 6-day-old PDA-plate cultures onto the injured site. Non-inoculated controls received three puncture wounds and 100 μL 0·1% Tween 20 in sterile water. Following inoculation, plants were individually covered with a plastic bag and moved into greenhouses – one set maintained at 24°C and another at 28°C. After 3 days, bags were removed and the uncovered plants held for an additional 7 days for disease development. Disease severity ratings (DSRs) were based on average reactions of three plants, on a scale of 0–4, where 0 = no visible symptom; 1 = necrotic lesion ≤ 2 mm in diameter; 2 = necrotic lesion > 2 mm in diameter; 3 = necrotic lesion >2 mm in diameter with water-soaked or yellowed margins; 4 = entire leaf yellow or dead. Reisolations on PCNB medium (Nash & Synder, 1962) were attempted from all isolate/plant-inoculation combinations to assess pathogenicity to orchids other than Phalaenopsis. The experiment was conducted twice.

Pathogenicity tests of F. solani isolates from other hosts to Phalaenopsis

Thirteen F. solani isolates, including one (CYP-01c) from Phalaenopsis, five (FS-3, FS-4, FS-5, FS-6 and FS-8) from pea, three (FS-53, FS-54 and FS-55) from chrysanthemum and three (FS-61, FS-64 and FS-65) from melon were evaluated for their pathogenicity to Phalaenopsis. Leaf laminas of Phalaenopsis cv. V3 were surface-sterilized by wiping with cotton saturated with 75% ethanol and injured by making three puncture wounds with a sterile needle. Inoculation was performed by two methods: (i) applying conidial suspension as described in the previous section or (ii) placing a 3-mm-diameter agar plug cut from a 7-day-old PDA-plate culture on the leaf surface at the injured site. Non-inoculated controls received three puncture wounds and 100 μL water or an agar plug cut from a sterile PDA plate. Inoculated plants were covered individually with plastic bags and held in a greenhouse at 28°C. After 3 days, the bags were removed and plants were held uncovered for another 7 days prior to DSR assessments. DSRs following conidial inoculation were on a scale of 0–4 as described in the previous section. Disease development following agar-plug inoculation was scored as with (+) and without (−) symptoms.

Pathogenicity of F. solani isolates from pea, chrysanthemum and melon to their respective hosts was confirmed by root-drench inoculation of 2-week-old seedlings with 5 mL of a suspension of 105 conidia mL−1. Inoculated plants were covered individually with plastic bags for 3 days and kept in the greenhouse. After removal of plastic bags, plants were kept in the greenhouse for an additional 30 days and then assessed for disease incidence. The pathogens were re-isolated to confirm Koch’s postulates. There were three replicates plants for each treatment and the experiment was conducted twice.

Test for host specificity among F. solani isolates from Phalaenopsis and non-orchid plants

Four isolates of F. solani, including one each from Phalaenopsis (CYP-01c), pea (FS-3), chrysanthemum (FS-53) and melon (FS-57), were tested for pathogenicity to pea (cv. Taichung no. 11), melon (cv. Netted Melon) and cucumber (cv. Feng-yan). Seeds were surface-disinfested by soaking in 1% (v/v) NaOCl for 30 s, rinsed twice in sterile distilled water, and sown in BVB No. 4 propagation medium (Bas Van Burren B.V.) in 5-cm-diameter pots, one seed per pot. The pots were held in a greenhouse at 28°C for 10 days and six seedlings for each isolate/seedling combination were inoculated 14 days after sowing by drenching each pot with 5 mL of a suspension of 105 conidia mL−1. Seedlings drenched in the same manner with sterile water served as controls. Individual plants were assessed for disease development and scored based on disease incidence 30 days after inoculation. Reisolations were made from all isolate/seedling combinations that resulted in disease development to confirm the causal agent. The experiment was conducted twice with six replicate plants for each treatment in each experiment.

DNA extraction, PCR amplification and sequencing

Total DNA of mycelial mats from each isolate of F. solani (Table 1) was extracted with sodium dodecylsulfate (SDS) detergent lysis buffer, followed by phenol/chloroform extraction and precipitation in ethanol with sodium acetate (Sambrook & Russell, 2001). Fragments of the genes encoding internal transcribed spacers (ITS), intergenic spacers (IGS) and β-tubulin genes were amplified from each isolate by PCR using primer pairs ITS1/ITS4, NF2/NF4 and Fu-tubulin3/FSB2 and the conditions described by White et al. (1990), Chen et al. (2009, 2008), respectively. PCR products were first purified using the PCR Clean-Up kit (GeneMark). Products were then subjected to BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems) under the following conditions: 25 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. Cycle sequencing reaction products were purified by ethanol precipitation and then analysed with an abi prism 3100 automated sequencer (Applied Biosystems).

Molecular phylogenetic analysis of ITS, IGS and β-tubulin gene regions

Three DNA regions of the ITS, IGS and β-tubulin gene from each isolate of F. solani shown in Table 1 were amplified. Both forward and reverse nucleotides were sequenced and assembled; once completed the nucleotide bases were aligned using clustal x v.1.8 (Thompson et al., 1997). Further visual alignments were done in sequence alignment editor (Se-Al) v.2.0 (Rambaut, 2000). The aligned sequences were analysed together with outgroup sequences of Fusarium proliferatum (FJ157218) and F. oxysporum (FJ545244) in rDNA-ITS analysis, F. proliferatum (DQ831905) in rDNA-IGS analysis and F. ambrosium (AY780137) in β-tubulin gene analysis.

Phylogenetic analysis of the aligned sequences was done by distanced parsimony methods. The distance matrix for the aligned sequences was calculated using Kimura’s two-parameter method and analysed with the neighbour-joining (NJ) method (Kimura, 1980). Maximum parsimony analysis was also performed. Included in the analyses were positions with gaps as a fifth character, since they may be informative for inferring phylogenies. Reliability of the inferred trees was estimated by 1000 bootstrap resampling using the same program. Parsimony analysis was done by paup v.4.0b (Swofford, 2000) using the heuristic search option with 100 random stepwise-addition sequences to search for the most parsimonious tree. Bootstrap (Felsenstein, 1985) values were generated with 1000 replicate heuristic searches to estimate support for clade stability of the consensus tree using the same program.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Pathogenicity of F. solani isolates from Phalaenopsis to other orchids

Results of the inoculation experiment showed that all 10 isolates of F. solani from Phalaenopsis were pathogenic to this host, causing symptoms of severe leaf yellowing on cv. V3 (Table 2). DSRs of 3–4 at 24°C and 2–4 at 28°C were assessed for the 10 Phalaenopsis isolates. Oncidium sp., Dendrobium sp., Cattleya sp., Cy. hybridum, Cy. ensifolium and Cy. sinense plants inoculated with the same 10 isolates showed slight necrosis or no symptoms (DSR 1 or 0) following inoculation and incubation at 24 and 28°C (Table 2). Moreover, attempts to reisolate F. solani from Oncidium sp., Dendrobium sp., Cattleya sp. and Cymbidium spp. plants with a DSR of 1 were unsuccessful.

Table 2.   Pathogenicity of isolates of Fusarium solani originally isolated from Phalaenopsis to other orchids
IsolateaDisease severity ratingb
PhcOnDeCaCyhCyeCys
24°C28°C24°C28°C24°C28°C24°C28°C24°C28°C24°C28°C24°C28°C
  1. aIsolates of F. solani were collected from diseased Phalaenopsis plants in Taiwan (see Table 1).

  2. bDisease severity rating (DSR) for each isolate/host combination at each temperature is an average DSR of three plants. DSR scale was 0–4: 0 = healthy, no visible symptoms; 1 = necrosis ≤ 2 mm and confined to the inoculation site; 2 = necrosis > 2 mm; 3 = necrosis > 2 mm with water-soaked border or slight yellowing at the inoculation site; 4 = dead or completely yellowed leaf.

  3. cHost abbreviations are as follows: Ph: Phalaenopsis sp. cv. Sogo Yukidian; On: Oncidium sp.; De: Dendrobium sp.; Ca: Cattleya sp.; Cyh: Cymbidium hybridum; Cye: Cy. ensifolium; Cys: Cy. sinense.

CPY-01c43011111101111
CYY3c43010100101111
FM2b43011101111111
FN2a44010101111111
FN2c32011101111011
SJ2a44010101111110
SJ2b44011101111111
SLuY-01a43011100111110
SLuY-01c43011111111111
YS31a43111101111111

Pathogenicity of F. solani isolates from other hosts to Phalaenopsis

Results of the pathogenicity tests following inoculation by both conidial suspension and mycelial-disc methods showed that F. solani isolate CYP-01c from Phalaenopsis was very aggressive to this host, resulting in a DSR of 4 following conidial inoculation and positive symptom expression following mycelial-disc inoculation (Table 3). At the same time, isolates of F. solani from other hosts, including six from pea, three from chrysanthemum and four from melon failed to elicit visual symptoms in Phalaenopsis plants and thus were considered to be avirulent to Phalaenopsis (Table 3). Likewise, Phalaenopsis isolate CYP-01c was avirulent to pea, chrysanthemum, cucumber and melon (Table 4). Isolate FS-3 from pea was pathogenic only to pea plants, isolate FS-53 from chrysanthemum was pathogenic only to chrysanthemum and isolate FS-57 from melon was pathogenic only to melon and cucumber plants, all with 100% infection (Table 4).

Table 3.   Pathogenicity of isolates of Fusarium solani originally isolated from Phalaenopsis, pea, chrysanthemum or melon to Phalaenopsis sp. cv. Sogo Yukidian
Isolate sourceIsolateResponse by Phalaenopsis
Spore suspensionMycelial disc
  1. aDisease severity scale rated on a 0–4 scale: 0 = healthy, no visible symptoms; 1 = necrosis ≤ 2 mm and confined to the inoculation site; 2 = necrosis > 2 mm; 3 = necrosis > 2 mm with water-soaked or yellowed margins; 4 = leaf dead or completely yellowed.

  2. b+ symptoms expressed; − no symptoms.

  3. cPhalaenopsis leaves were punctured and received 100 mL sterile water.

P. amabilis cv. Han-BenCPY-01c4a+b
Pisum sativumFS-31
FS-41
FS-50
FS-60
FS-81
Chrysanthemum indicumFS-531
FS-540
FS-550
Cucumis meloFS-571
FS-610
FS-641
FS-650
Controlc 0
Table 4.   Pathogenicity of Fusarium solani isolates from Phalaenopsis, pea or melon towards pea, chrysanthemum, cucumber and melon
IsolateaDisease severity rating (%)b
PeaChrysanthemumCucumberMelon
  1. aIsolates of F. solani were: CYP-01c isolate from Phalaenopsis, FS-1 from pea, FS-53 and FS-57 from melon.

  2. bDisease severity was recorded 30 days after inoculation.

  3. cPlants were inoculated with sterile distilled water.

CYP-01c0·00·00·00·0
FS-31000·00·00·0
FS-530·01000·00·0
FS-570·00·0100100
Controlc0·00·00·00·0

Molecular phylogenetic analysis of ITS, IGS and β-tubulin gene of F. solani

The amplification products of the rDNA-ITS region including 5.8S rDNA of 35 isolates of F. solani was 526 base pairs (bp) long. The aligned and truncated rDNA-ITS sequences consisted of 573 characters, with 441 characters constant, 57 parsimony uninformative and 75 parsimony informative. An NJ tree constructed from the rDNA-ITS and 5.8S regions showed that the 35 isolates of F. solani from Phalaenopsis were in one group with 98% bootstrap values (Fig. 1). For maximum parsimony analysis, the result was similar to NJ analysis. Thus, the isolates of F. solani from Phalaenopsis fell into one group with 84% bootstrap values (data not shown). The ITS phylogram had a consistency index (CI) of 0·697, a retention index (RI) of 0·722, retention consistency (RC) of 0·503 and a tree length of 228.

image

Figure 1.  ITS and 5.8S rDNA sequence-based tree generated with neighbour-joining analysis. Numbers at branch modes indicate reliable values from bootstrap analysis with 1000 replications. Fusarium proliferatumFJ157218 and F. oxysporumFJ545244 were used as outgroups to root the tree. Ph Phalaenopsis; Cy = Cymbidium.

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The amplification products of the partial rDNA-IGS region of the 35 isolates of F. solani from Phalaenopsis were 443∼445 bp long. The aligned and truncated rDNA-IGS sequences consisted of 473 characters, with 300 characters constant, 88 parsimony uninformative and 85 parsimony informative. The NJ tree constructed from the partial rDNA-IGS region showed that the 35 isolates of F. solani from Phalaenopsis fell into a single group with 100% bootstrap values (Fig. 2). For maximum parsimony analysis, the result was the same as the result of NJ analysis, in which all the 35 isolates of F. solani from Phalaenopsis fell into one group with 100% bootstrap values (data not shown). The rDNA-IGS phylogram had a consistency index (CI) of 0·879, a retention index (RI) of 0·907, retention consistency (RC) of 0·798 and a tree length of 290.

image

Figure 2.  IGS rDNA sequence-based tree generated with neighbour-joining analysis. Numbers at branch modes indicate reliable values from bootstrap analysis with 1000 replications. Fusarium oxysporumEF661647 was used as an outgroup to root the tree. Ph = Phalaenopsis; Cy = Cymbidium.

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The amplification products of the partial β-tubulin gene resulted in a fragment of 814∼816 bp for the 35 isolates of F. solani from Phalaenopsis. The aligned and truncated partial β-tubulin sequences consisted of 1044 characters, with 987 characters constant, 26 parsimony uninformative and 31 parsimony informative. The NJ tree constructed from partial β-tubulin sequences showed that the 35 isolates of F. solani from Phalaenopsis form a single group with 100% bootstrap values (Fig. 3). For maximum parsimony analysis, the result was similar to that of NJ analysis, as the isolates of F. solani from Phalaenopsis fell into one group with 100% bootstrap values (data not shown). The β-tubulin sequence phylogram had a consistency index (CI) of 0·951, a retention index (RI) of 0·985, retention consistency (RC) of 0·937 and a tree length of 61.

image

Figure 3. β-tubulin gene sequence-based tree generated with neighbour-joining analysis. Numbers at branch modes indicate reliable values from bootstrap analysis with 1000 replications. Fusarium ambrosiumAY780137 was used as an outgroup to root the tree. Ph = Phalaenopsis; Cy = Cymbidium.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fusarium solani has been reported as the pathogen causing leaf yellowing or root rot of Phalaenopsis (Morita et al., 1992; Kim et al., 2002; Lin et al., 2007; Chen et al., 2008, 2009), dry rot or root rot in Cymbidium (Benyon et al., 1996; Ichikawa & Saito, 1998; Lee et al., 2002) and stem rot or root rot of Dendrobium (Latiffah et al., 2008, 2009). However, the pathogenicity of F. solani on different orchids is not clear. The results of the present study demonstrated that the isolates of F. solani from Phalaenopsis are highly host specific (Table 2). Moreover, the disease survey of Phalaenopsis in greenhouses indicated that F. solani was the major pathogen causing leaf yellowing and root rot (Chen et al., 2008). Pathogenic tests in the present study showed that isolates of F. solani from Phalaenopsis caused little or no symptoms in Cymbidium spp., Oncidium sp., Cattleya sp. and Dendrobium sp. Furthermore, F. solani could not be re-isolated from tissue expressing limited symptoms. Although F. solani has been shown in other studies to cause disease in Cymbidium (Benyon et al., 1996; Ichikawa & Saito, 1998; Lee et al., 2002) and Dendrobium (Latiffah et al., 2008, 2009), there are no data to demonstrate pathogenicity of F. solani from these orchids to Phalaenopsis. Thus, the host ranges of F. solani that attack Cymbidium and Dendrobium are not clear. There is no formal report of a disease caused by F. solani in Cymbidium or Dendrobium in Taiwan. This is the first study to demonstrate that F. solani isolates from Phalaenopsis are not pathogenic to Cymbidium, Oncidium, Cattleya and Dendrobium.

Other than Phalaenopsis, 12 plants are recorded in Taiwan as hosts of F. solani, viz. Allium sativum, Althaea rosea, Arachis hypogaea, Ardisia crenata, Citrus sinensis, Cosmos sulfurous, Cucumis melo, Passiflora edulis, P. sativum, Solanum tuberosum, Catharanthus rosea and Syzygium samarangense (Sun & Huang, 1997; Hsu et al., 2002; Chen et al., 2006, 2008, 2009; Wang et al., 2010). Among the above-mentioned diseases incited by F. solani, only two ‘formae speciales’, F. solani f. sp. pisi and F. solani f. sp. cucurbitae, have been confirmed by pathogenicity tests (Sun & Huang, 1997; Chen et al., 2006). In the present study, isolates of F. solani from Phalaenopsis did not incite disease in pea, cucumber or melon. Similarly, the isolates of F. solani from pea, melon and chrysanthemum failed to incite disease in Phalaenopsis, although some isolates caused necrotic hypersensitive-like reactions (Table 3). The results show that isolates of F. solani from Phalaenopsis are highly host-specific to Phalaenopsis.

The phylogenic analysis of rDNA-ITS showed that the isolates of F. solani from Phalaenopsis form a monophylogenic group. The rDNA-ITS region has been used for phylogenetic analysis of the F. solani complex and distinguishing different ‘formae speciales’ (Suga et al., 2000). The rDNA-ITS sequence data from the present study show isolates of F. solani from Phalaenopsis to be a tight-knit group and distinct from other F. solani. Moreover, rDNA-IGS and β-tubulin gene regions clearly distinguish Phalaenopsis isolates from other F. solani isolates. Previous studies validated the potential of the rDNA-IGS region to separate ‘formae speciales’ such as F. oxysporum f. sp. lactucae (Fujinaga et al., 2005) and F. oxysporum f. sp. lycopersici (Kawabe et al., 2005), and the β-tubulin gene has been used to distinguish morphologically obscure species (Chung et al., 2006). Molecular phylogenetic analyses were successfully used in the present study to show that isolates of F. solani from Phalaenopsis are significantly different from other pathogenic F. solani. Results of pathogenic tests and molecular characterizations suggest that the isolates of F. solani from Phalaenopsis represent a new ‘forma specialis’, which it is proposed be designated F. solani f. sp. phalaenopsis.

Three isolates of F. solani (CymFS-1, CymFS-2 and CymFS-3) from roots of Cy. ensifolium showing slight necrosis were included in the molecular phylogeny study. Results showed that the three F. solani isolates from Cy. ensifolium are located in the same molecular group as those from Phalaenopsis (Figs 1–3). Cymbidium spp. have been reported as a host of F. solani causing a dry rot (Benyon et al., 1996; Ichikawa & Saito, 1998; Lee et al., 2002). Although inoculation tests with the three Cymbidium isolates of F. solani showed them to be pathogenic to Phalaenopsis sp. cv. V3, causing leaf yellowing, they failed to incite symptoms in Cymbidium spp. (data not shown), suggesting that the isolates recovered from Cymbidium are F. solani f. sp. phalaenopsis isolates that are able to survive on but are not pathogenic to Cymbidium.

Whether previously reported isolates of F. solani from Phalaenopsis, Cymbidium and Dendrobium also belong to f. sp. phalaenopsis remains to be investigated. In this study, the isolates of F. solani obtained from Phalaenopsis in Taiwan were homothallic and able to produce asci and ascospores identified as Haematonectria haematococca. The teleomorph stage of the F. solani obtained from Cymbidium (Benyon et al., 1996; Ichikawa & Saito, 1998; Lee et al., 2002) and Dendrobium (Latiffah et al., 2008, 2009) was not described and characterized. On the other hand, isolates of F. solani causing disease on Phalaenopsis in China (Lin et al., 2007), Japan (Morita et al., 1992) and Korea (Kim et al., 2002) did produce asci and ascospores, just like the isolates in Taiwan, but their pathogenicity to other orchids needs to be tested.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We sincerely thank Dr Lowell L. Black, Plant Pathologist, Monsanto Vegetable Seeds, DeForest, WI, USA for critical review of this manuscript. This research was funded by projects 96AS-14.4.1-BQ-B2, 97AS-14.4.1-BQ-B2 and 98AS-9.4.1-BQ-B2 from the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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