ISBN : 978-602-8915-93-9 OCCURENCE OF BACTERIAL SOFT ROT OF PHALAENOPSIS ORCHIDS IN YOGYAKARTA AND WEST JAVA, INDONESIA Tri Joko1, Dwi Kiswanti1, Hanudin2, Siti Subandiyah1 Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia 2 Indonesian Ornamental Crops Research Institute, Segunung, Cianjur, West Java 1
ABSTRACT A soft rot disease was observed on Phalaenopsis sp., in major growing areas of Yogyakarta and West Java, Indonesia during April-July 2010. Typical symptoms were soft rots characterized by moist and watery decay of the one or whole leaves, which initiated as small water-soaked lesions and enlarged rapidly to the entire leaves. Among surveyed areas, field observations found that disease occurred on all sampling areas with the maximum disease incidence and disease intensity (100% and 46.2%, respectively) were recorded from Kulon Progo and Sleman. The causal organism isolated from the infected leaves was identified as Dickeya sp. (Syn. Erwinia chrysanthemi) and Pseudomonas sp. based on physiological and biochemical characteristics and confirmed by PCR test. Pathogenicity of the bacteria was proven by Koch's postulations. Artificial inoculation of the bacteria produced the same soft rot symptoms on the Phalaenopsis leaf, from which the same bacterium was isolated and identified. Keywords : Phalaenopsis, bacterial soft rot, identification
INTRODUCTION Phalaenopsis, the moth orchid, is perhaps the most preferred and famous in the trade, through the development of many artificial hybrids. The generic name means "Phalaen[a]-like" and is probably a reference to the genus Phalaena, the name given by Carolus Linnaeus to a group of large moths; the flowers of some species supposedly resemble moths in flight. For this reason, the species are sometimes called moth orchids (Anonim, 2011). During the last few years orchids sales has been increasing with value approximately $100,000,000 in USA, and over 75% of all orchids sold are Phalaenopsis (Griesbach, 2002). Large scale, potted Phalaenopsis production is occurring in the Netherlands, Germany, China, Taiwan, United States, and Japan. Moth orchids are the most popular orchids and are very easy to grow in most homes with high humidity being the most difficult growing requirement to meet (Anonim, 2003). In India, species of the genus Phalaenopsis are the most important group among the other wellknown species of orchids popular with growers all over the world (Pradan, 2005). Between 40 and 45 species: from India to S China, Thailand, Indochina, Malaysia, and Indonesia to the Philippines and New Guinea, the majority in Indonesia and the Philippines; 12 species (four endemic) in China. The genus Phalaenopsis has been monographed by Christenson (Xinqi and Wood, 2009).
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ISBN : 978-602-8915-93-9 In Indonesia, the beauty of this genus member (P. amabilis) has made it promoted as “Puspa Pesona”, one of three national flowers (Anonim, 2011). Diseases affecting the family Orchidaceae include root diseases, stem and pseudobulb decays, leaf spots, and flower blights. Worldwide, one of the most common and important limiting factors for cultivating a Phalaenopsis orchid is soft rot disease that attack many nurseries all over the world (Keith et al., 2005). Small water-soaked spots appear on the leaves and often are surrounded by yellow halos. The infection will rapidly rot the leaves and roots and spread more slowly into the rhizomes or pseudobulbs. This wet rot may have a foul odor and has a water soaked appearance. In Phalaenopsis, the disease spreads so rapidly that plants may be completely rotted in 2-3 days. The bacterial pathogens are opportunistic organisms that can enter through wounds (Bottom, 2011). The soft-rot pathogen is armed by coordinated production of high levels of multiple exoenzymes, including pectinases, cellulases, and proteases, which break down plant cell walls and release nutrients for bacterial growth (Toth et al. 2003). The pectolytic and other macerating enzymes produced by soft-rot pathogens result in a loss of structural integrity in host tissue and a characteristic soft-rot (Choter and Sivasithamparam, 1983; Goto, 1992). Soft-rot bacteria have developed a complete and efficient set of enzymes and isoenzymes to deal with the complexity of plant cell wall polymers. The major pathogenicity determinants of these bacteria are plant cell wall degrading enzymes such as pectinases (pectate lyase, polygalacturonase, pectin methyl esterase), cellulases, and proteases (Collmer and Keen, 1986). Among these enzymes, pectinases are considered to be the main exoenzymes which break down and utilize pectins in the middle lamella and plant cell walls, causing tissue collapse, cell damage and cell leakage (Barras et al., 1994; Hugouvieux-Cotte-Pattat et al., 1996). In this study, we described the isolation of soft rot bacteria on Phalaenopsis sp. in Yogyakarta and West Java. Characterization was done either by physiological and biochemical studies or by molecular studies using PCR technique.
MATERIALS AND METHODS Disease occurence and symptoms. Observation of the occurrence of a soft leaf rot on the Phalaenopsis orchids was done in major growing areas of Yogyakarta and West Java, Indonesia during April-July 2010. The survey and sampling of diseased orchids were conducted in Yogyakarta district, Kulon Progo district, Sleman district, Bogor district, Lembang district, and Cianjur district. These areas were selected as surveyed places because of being known as orchid growing areas in Yogyakarta and West Java (Tabel 1). It was also commonly reported the insidence of the soft rot diseases from these areas.
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Tabel 1. Surveyed areas of Phalaenopsis sp. showing soft rot symptom Province District Kulon Progo DI Yogyakarta Sleman Yogyakarta Bogor West Java Lembang Cianjur Assessment of disease occurence. At least two orchid nurseries, as representative district sampling area of each province, were surveyed since April - July 2010. Fifty posts of orchids plants were randomly sampled from each surveyed nursery. Disease incidence (DI) was calculated by the following equation (Cooke, 2006): No. of infected plant units DI = ---------------------------------------- ×100 Total no. of plant units assessed Meanwhile, the disease intensity (I) was determined according to alternative rating scale proposed by Handayati et al. (2004) in which scale 0 = no symptom, scale 1 = small spot with 1% of severity level on infected leaf, scale 2 = 2–10%, scale 3 = 11–25%, scale 4 = 26–50% and scale 5 = >50% of leaf, respectively and then measured using the equation proposed by Norman et al. (1977) as follows: ∑(nv)
I=
ZN
Σ (n × v) = Sum of the symptomatic plant and their corresponding score scale N = Total number of sampled plant Z = Highest score scale
Bacterial isolation and pathogenicity. Pathogen isolation was conducted using infected Phalaenopsis leaves of surveyed area in Yogyakarta and West Java. Small leaf tissues were cut from the edge of the diseased area with flame-sterilized razor blade and surface-sterilized with 75% ethanol for 30 sec and 1% sodium hypochlorite for 60 sec, and rinsed with sterile water. The cut leaf tissues were placed on a flamed glass tube containing 5 ml of sterile water, allowing 15 min to stand, which were streaked on yeast peptone agar (YPA). Single bacterial colonies formed after 2-3 days of incubation at 28C were isolated from the culture plates, and stored in 30% gycerol at -20C for further analysis (Fahy and Hayward, 1983; Schaad et al., 2001). One isolate from several colonies with the same colony morphology (forming grayish-white colonies on nutrient agar) was selected as a type culture for further studies. The bacterial isolate grown on nutrient agar for 2-3 days was collected in ISNAR C2F PROCEEDING Natural Resources Climate Change and Food Security in Developing Countries Surabaya, Indonesia, June 27-28, 2011
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ISBN : 978-602-8915-93-9 sterile distilled water and the concentration was adjusted to 1.0×108 cells/ml as inoculum for pathogenicity test. One-year-old leaves of Phalaenopsis sp. in a pot were inoculated with the bacterial suspension after wounding with a sterilized needle until run-off, and attached with moistened cotton to allow enough moisture, which were kept at 28C in an plastic bag. The experiment was replicated three times, and sterile distilled water was used as control. Identification of bacterial isolates. Bacterial characteristics of the isolates from Phalaenopsis sp. were investigated by the methods of Dickey and Kelman (1988). Gram staining, colony color on nutrient glucose agar (NGA), fluorescent pigment production on King's B agar, anaerobic growth, growth on osmotic condition (5% NaCl), growth on various temperature (10C, 20C, 37C). The morphology of the bacterial cells was investigated with the aid of light microscope connected to computer (Opti Lab). Polymerase Chain Reaction (PCR) identification. Bacterial genomic DNA was extracted from either bacterial suspensions in ddH2O (108 cells ml−1), prepared from 24 h cultures in YP medium, that was centrifuged for 2 min at 12,000×g. The DNA was extracted and purified using Phenol-Chloroform extraction methods (Ausubel et al., 1990). PCR was performed in 20 μl reaction volumes containing 10 μl of Master Mix (MMR, Microzone), 1 μl of 10 pmol each of the Pectobacterium-specific primer pectate lyase-encoding gene cluster, Y1/Y2 (Darrasse et al. 1994), Dickeya-specific primer, ADE1/ADE2 (Nassar et al. 1996), and Pseudomonas-specific primer, fPs16S/rPs23S (Locatelli et al. 2002), 1 μl of bacterial genomic DNA, and 17 μl of ddH2O. DNA amplification was performed using a Termocycle PCR machine. PCR conditions consisted of 2 min at 94°C, followed by 35 cycles of 45 s at 94°C and 45 s at 72°C, 2 min at 72°C, and a final extension at 72°C for 3 min (PCR product size 450 bp). Amplified DNA fragments (12.5 μl) were run on a 1.5% agarose gel at 80 V for 1 h and visualised under UV light following ethidium-bromide staining (Joko et al., 2007) .
RESULTS AND DISCUSSION
Disease occurence and symptoms. Occurence of disease plant was observed often in most of surveyed area in Yogyakarta and West Java. Soft rot symptoms were characterized by moist and watery decay of the Phalaenopsis leaf. Initially one or several small water-soaked lesions were formed on a leaf, and enlarged rapidly to finally rot the whole leaf (Fig. 1A). Soft rot could be also initiated from shoot and enlarged to the whole plant causing rotting death (Fig. 1B).
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A
B
Fig. 1. The symptoms of soft rot disease of Phalaenopsis on leaf (A) and shoot (B), small water-soaked lession enlarged to either the whole leaf or plant.
Assessment of disease occurrence. A total of 9 orchid nurseries and small house yard in Yogyakarta and West Java had been successfully surveyed since April until July 2010. The disease occurred in 7 surveyed area with maximum disease incidence and disease intensity (100% and 46.2%, respectively) were recorded from Kulon Progo and Sleman (Table 2). Table 2. Occurence of soft rot disease sampled from Yogyakarta and West Java Location (District) Disease Incidence (%) Disease Intensity (%) Kulon Progo 100 8.1 Sleman 0.6 46.2 Yogyakarta 37.5 10.9 Bogor 5 41.85 Lembang 35 16.9 Cianjur 20 24,8
Bacterial isolation and pathogenicity. Single bacterial colonies formed after 2-3 days of incubation at 28C were isolated from the culture plates, and stored in 30% gycerol at -20C for further use. One isolate from several colonies with the same colony morphology (forming grayish-white colonies on nutrient agar) was selected as a type culture for further studies. The selected isolates after purification from single colony was shown in Table 3. We found 14 isolates caused typical soft rot symptoms on the leaves of Phalaenopsis sp. at 12 hrs after artificial wound inoculation (Fig. 2), showing watery decay of the whole stem within 12 hours after the bacterial inoculation. Waterinoculated control did not induce any soft rot symptoms.
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Fig. 2. Pathogenicity assay of the isolates on leaves of Phalaenopsis sp.
Table 3. Selected isolate of soft rot bacteria from Yogyakarta and West Java No Isolate Orchid species District 1. Ph Kp Phalaenopsis sp. Kulon Progo 2. Ph Yk Phalaenopsis sp. DIY 3. Ph Sl2 Phalaenopsis sp. Sleman 4. Ph Sl2 Rd2 Phalaenopsis sp. Sleman 5. Pf Agr Phalaenopsis sp. Cianjur 6. Ph 7 Phalaenopsis sp. Cianjur 7. Pal 3.4 Phalaenopsis sp. Cianjur 8. Pal 1.2 Phalaenopsis sp. Cianjur 9. Pal 3.2 Phalaenopsis sp. Cianjur 10. Pal 2.2 Phalaenopsis sp. Cianjur 11. Ph Lb1 Phalaenopsis sp. Lembang 12. Ph Lb2 Phalaenopsis sp. Lembang 13. Ph Cis Phalaenopsis sp. Cisarua 14. Ph Dh Phalaenopsis sp. Sleman
Identification of bacterial isolates. The 14 isolates were Gram-negative and formed grayish white colonies at 2 days after incubation on yeast peptone agar. Several isolates did not produce fluorescent pigments on King’s B agar and could grow aerobically or anaerobically (Table 3). Microscopic observation exhibited that the bacterial cells were bacilli form (Fig. 3). These physiological and morphological characteristics of the several bacterial isolated coincide with those of the genus Erwinia (Dickey and Kelman, 1988), but some did not such as production of fluorescent pigment that was a characters of Fluorescent pseudomonads (Scarpellini et al., 2004). Several isolates showed positive reactions on the tests oxidatif fermentatif, and grew at 10C, 20C, 37C, but some did not. All of the isolates showed bacilli form.
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ISBN : 978-602-8915-93-9 Table 3. Physiologi and biochemical characters of the isolates No.
Isolate
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Ph Kp Ph Yk Ph Sl2 Ph Sl2 Rd2 Pf Agr Ph 7 Pal 3.4 Pal 1.2 Pal 3.2 Pal 2.2 Ph Lb1 Ph Lb2 Ph Cis Ph Dh
Oxidatif/ fermentatif F O + + + + + + + + + + + + + + + + + + + + + + + +
Growth at 5% NaCl + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ -
Florescence
+ + + +
Temperature Growth 10C + + + + + + + + + + + +
20C + ++ ++ +++ ++ +++ + ++ ++ ++ ++ +++ + +
37C ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
Shape
Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli
Fig. 3. Bacilli form of all of the isolates of soft rot bacteria
Molecular Identification. The expected 420 bp PCR-amplified fragment, corresponding to the conserved regions of the Dickeya sp. pectate lyase-encoding gene cluster (pelADE; Nassar et al. 1996), was obtained with l strains tested (Fig. 4), no PCR products were obtained for Pectobacterium sp. using Y1/Y2 specif primer (Fig. 5). In an additional analysis, the expected 1200 bp PCR-amplified fragments, corresponding to the conserved 16S-23S ITS (ITS-PCR) regions of Pseudomonas sp., were obtained for all 1 tested (Fig. 6).
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M1 1
2
3
4
5
6
7
8
9 10 11 12 13 14 M2
bp
bp
1000
1000
500
500 400
Fig. 4. Detection of the isolates using primer Ade1/Ade2
M1 1 2
3
4
5
6
7
8
9 10 11 12 13 14 M2
bp
bp 1000
750 500
500
Fig. 5. Detection of the isolates using primer fPs16S/rPs23S
M1 1
2
3
4
5
6
7
8
9 10 11 12 13 14 M2 bp
bp 1000
1000
500
500 400
Fig. 6. Detection of the isolates using primer Y1/Y2
ACKNOWLEDGEMENT We thank Ministry of Agriculture, Republic of Indonesia for funding this research through KKP3T research project.
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