Abstract: Knowing the unfavorable environment for the growth of a pathogen can be utilized as the basic information in developing appropriate strategies to prevent disease occurrence on dragon fruit. Several environmental factors including temperature, pH and salinity, as well as biotic factor including three antagonistic bacteria species, namely Bukholderia cepacia, B. multivorans and Pseudomonas aeruginosa against Bipolaris sp., Colletotrichum gloeosporioides, Botryosphaeria sp. and Monilinia sp., were investigated. Mycelial growth of all tested fungi was constantly inhibited by a temperature of 35°C, while a temperature of 25°C was quite suitable for their growth. A temperature of 30°C was favorable for the growth of Colletotrichum gloeosporioides. Under different pH condition, the growth of tested fungi was mostly inhibited by extreme pH of 4 and 10. The salinity assay showed that Monilinia sp. was not affected by all treatments among tested fungi. Only concentration 100 ppm could reduce the growth of Bipolaris sp., though its inhibition statistically affected on 4 and 6 Days after Incubation (DAI). Meanwhile, the in vitro examination of antagonistic bacteria resulted in Bukholderia multivorans which was highly effective in inhibiting the growth of examined fungi, except Monilinia sp., which was more significantly influenced by B. multivorans and B. cepacia. The proper combination of environmental modification may be useful for the growth of crop in the field as well as the storage life of the fruit at postharvest preservation.
INTRODUCTION
Dragon fruit (Hylocereus spp.) is a vine climbing cactus species which originated from South America (Crane and Balerdi, 2005). This crop is characterized by a skin covered with dragon-like scales. It was firstly introduced in large scale into Malaysia about two decades ago by Golden Hope Company locating at Sungai Wangi Estate (Perak). Furthermore, at the beginning of 1999, the commercial cultivations were then developed in Kluang (Johor), Kuala Pilah (Negeri Sembilan) and Sitiawan (Perak) (Halimi and Satar, 2007).
Many records of diseases on dragon fruit have been documented from several white-fleshed and yellow species-producing countries. Some earlier studies have successfully recognized a number of fungal species causing various diseases, such as Alternaria sp., Ascochyta sp., Aspergillus sp., Bipolaris cactivora, Botryosphaeria dothidea, Capnodium sp., Colletotrichum gloeosporioides, Dothiorella sp., Fusarium sp., Gloeosporium agaves, Macssonina agaves, Phytopthora sp. and Sphaceloma sp. (FAO, 2004; Sijam et al., 2008; Le Bellec et al., 2006; Palmateer et al., 2007; Paull, 2007; Taba et al., 2006, 2007; Valencia-Botin et al., 2003; Wang and Lin, 2005). In Peninsular Malaysia, several pathogenic fungi on this crop, such as Bipolaris sp., Botryosphaeria sp., C. gloeosporoides and Monilinia sp., causing fruit and stem end rot, brown spot, anthracnose and fruit brown rot are found on this crop, respectively (Masyahit et al., 2008, 2009).
Knowledge of the basic biology of the pathogen, such as germination and sporulation in relation to environmental factors, is useful in the development of a more sustainable strategies of disease management (Xu et al., 2001). It is necessary to understand the precise environmental conditions for infection and disease development in order to determine the appropriate time of fungicide application and perhaps the implementation of alternative disease control measures (Percich et al., 1997). This study assessed the environmental factors as well as antagonistic bacteria against isolated fungal pathogens of dragon fruit under in vitro conditions.
MATERIALS AND METHODS
The experiments were carried out in the Microbiology Laboratory, Department of Plant Protection, Faculty of Agriculture, University Putra Malaysia, during February-March 2009. Fungal isolates used in this experiment were sub-cultured on PDA (Oxoid Ltd., Basingtoke, Hampshire; England) from isolate collection of our two earlier studies (Table 1). Some environmental factors including temperature, pH and salinity and three antagonistic bacteria species, namely Bukholderia cepacia, B. multivorans and Pseudomonas aeruginosa against those pathogenic fungi were investigated.
Effect of temperature: Fungal isolates were cultured on PDA plates and then incubated at 4 different temperatures, 20, 25, 30, or 35°C for 10 days according to a slight modification of the method developed by Baird (2004). Fungal growth was monitored by measuring the mycelial diameter using a digital caliper (Digimatic Caliper; Mitutoyo Corporation, Japan) every two days. The treatments were replicated three times.
Effect of pH: Fungal isolates were cultured on PDA. pH had been adjusted to 4, 5.5, 7, 8.5 or 10. The pH was adjusted by dropping 1 N HCl for decreasing pH and 1 N NaOH for increasing pH into autoclaved-PDA media before cooling according to the method developed by Fayzalla et al. (2008). pH was determined using Delta 320 pH meter (Mettler, Toledo). Fungal growth was monitored by measuring the mycelial diameter using digital caliper every two days. The treatment of this experiment was replicated three times.
Effect of salinity: Fungal isolates were cultured on PDA plates for 10 days with five different concentrations of salinity, i.e., 0, 1, 10, 100, 1000 mg L-1, which were adjusted by adding NaCl (Sharlau Chemie S.A., Barcelona, Spain) before autoclaving following the procedure developed by Al-Rokibah et al. (1998) with a little modification. Fungal growth was monitored by measuring the mycelial diameter using digital caliper every two days. Treatments were replicated three times.
| Table 1: | Fungi isolated from diseased dragon fruit used in this experiment |
Effect of antagonistic bacteria: This experiment was conducted with dual culture procedure following method developed by Sijam and Dikin (2005) by culturing both fungal isolate and antagonistic bacterium in PDA plates for 10 days at room temperature. Antagonistic bacteria, i.e., Bukholderia cepacia, B. multivorans and Pseudomonas aeruginosa, were sub-cultured on NA (Oxoid Ltd., Basingtoke, Hampshire; England) from collection of Microbiology Laboratory, Department of Plant Protection, Faculty of Agriculture, University of Putra Malaysia. A plug of fungal isolate (4 mm in diameter) was placed 2 cm from the edge of the 9 cm plate; while the antagonistic bacterium was line-streaked on 2 cm from other edge of the same plate. Fungal growth was monitored by measuring both the mycelial radial toward the margin of plate (considered as Ro) and that straight the bacterial streak (considered as Rt) using digital caliper every 2 days. The treatment of this experiment was replicated three times. Zone inhibition of antagonistic bacteria against fungal isolates was calculated with this following formula:
| PI | = | Percentage of inhibition (%) |
| Ro | = | Mycelial growth of fungi toward margin plate (mm) |
| Rt | = | Mycelial growth of fungi toward the bacterial streak (mm) |
Statistical analysis: These in vitro experiments were employed with Completely Randomized Design (CRD). Following the statistical procedures developed by Gomez and Gomez (1984), data were analyzed with ANOVA under Duncan Multiple Range Test (DMRT) using SAS® System for Windows V8 software (SAS Institute Cary, North California; USA).
RESULTS AND DISCUSSION
Effect of temperature: Overall, test of temperature treatment showed that the mycelial growth of the fungi under test was constantly inhibited by a temperature of 35°C; while temperature 25°C was quite suitable for their growth. A temperature of 30°C was favorable for the growth of C. gloeosporioides. The mycelial growth of Bipolaris sp., Botryosphaeria sp. and Monilinia sp., was more encouraged by temperature 20 and 25°C for 10 days incubation (Fig. 1a, c and d) while temperatures range of 25-30°C was optimum for the growth of C. gloeosporioides in this study (Fig. 1b).
Effect of pH: The in vitro assay of pH effect generally revealed that the growth of tested fungi was mostly inhibited by extreme pH, i.e., pH 4 and pH 10. The inhibition of pH 4 was effective until last incubation, except for Monilinia sp. which was only influenced at 2 Days after Incubation (DAI); whilst pH 10 was significant for 6 days incubation. At 6 days after incubation, growth of the two fungal species, Bipolaris sp. and Botryosphaeria sp., were more significantly repressed than others. In the meantime, pH values at range 5.5-8.5 were less effective against the growth of tested fungi. The effect of pH treatment of 5.5-10 in range decreased after the fourth observation (8 DAI) (Fig. 2a-d).
Effect of salinity: The findings of salinity assay described that Monilinia sp., was not affected by all treatments among tested fungi. For this fungus, treatments were only significant at 0.05 level in inhibiting its growth at first observation and it then could reach optimum growth only at 4 DAI. The significant effect of inhibition against C. gloeosporioides was only shown until 6 days incubation, while those entire treatments had highly significant different effects on Bipolaris sp., at 4 DAI on the 0.01 level. All salinity concentrations did not significantly inhibit the growth of Botryosphaeria sp. Only concentration 100 ppm could reduce the growth of Bipolaris sp., though its inhibition statistically affected on 4 and 6 DAI (Fig. 3a-d).
Effect of antagonistic bacteria: The results of antagonistic bacteria test highlighted that Bukholderia multivorans was highly effective in inhibiting the growth of examined fungi, except Monilinia sp., which was more significantly influenced by B. cepacia. It was found that Pseudomonas aeruginosa was only more effective at 4, 6 and 10 DAI against Bipolaris sp., rather than others. The ability of tested bacteria in inhibiting the growth of Bipolaris sp., (Fig. 4a) and C. gloeosporioides (Fig. 4b) fungi was mostly optimum at 8 DAI and fluctuated against Botryosphaeria sp., with the peak at last incubation (Fig. 4c). Meanwhile, only B. multivorans gradually increased in inhibiting Monilinia sp. with the optimum at 8 DAI; while the inhibition percentage of others was decreased until the last incubation (Fig. 4d).
In general, the growth of tested fungal pathogenic in this study was significantly inhibited by the extreme (minimum and maximum) level of treatments, except for salinity treatment. Meanwhile, three antagonistic bacteria used could increasingly restrict the mycelial growth of tested pathogen, excluding against Monilinia sp. These findings, however, were unlike those examining other species of the same fungi on different crops. Maximum proportional germination of Bipolaris sorokiniana causing disease complex on Poa pratensis and Agrostis palustris was greatest at 25 and 30°C, respectively, but it failed to germinate at 35°C (Hodges 1975); while Barba et al. (2002) observed that the sporulation of B. sorokiniana on barley seed reached its maximum at 19.3°C.
Some slight different ranges of the most favorable temperatures were also found on other Botryosphaeria species. Pycnidial formation of Botryosphaeria ribis was induced either under continuous illumination at 21°C favoring development of uniloculate pycnidia or in alternating light (12-27°C) and darkness (12-21°C) enhancing formation of multiloculate pycnidia (Smith and Fergus, 1971).
| Fig. 1: | The effect of temperature against (a) Bipolaris sp. (b) Colletotrichum gloeosporioides (c) Botryosphaeria sp. and (d) Monilinia sp., under in vitro condition |
| Fig. 2: | The effect of pH level against (a) Bipolaris sp. (b) Colletotrichum gloeosporioides (c) Botryosphaeria sp. and (d) Monilinia sp., under in vitro condition |
| Fig. 3: | The effect of salinity against (a) Bipolaris sp. (b) Colletotrichum gloeosporioides (c) Botryosphaeria sp. and (d) Monilinia sp., under in vitro condition |
Different optimum temperatures were even observed within Monilinia species by some authors on their previous studies. The rate of M. fructicola conidial growth on fresh stone fruits in incubators were maximum at 15°C and minimum at 25°C (Phillips, 1984); whereas like this current study, the conidia of M. fructigena causing brown rot disease on apple and pear fruit in UK had already germinated only 2 h after seeding on the plates at 20 and 25°C, about 48 and 55%, respectively and then declined at 30°C (Xu et al., 2001).
Meanwhile, temperature range of 25-30°C for optimum growth of C. gloeosporioides obtained in this study was slightly similar to those observed on earlier experiments on same fungus infecting other crops. Those works reported that a temperature range of 20-30°C as the optimum temperature for the lesion development on rubber leaves (Wastie, 1972), in vitro disease development on Stylosanthes sp. (Irwin et al., 1984) as well as germination and production of conidia (Fitzell and Peak, 1984), growth and sporulation (Davis et al., 1987), appressoria production (Estrada et al., 2000) and infection development (Akem, 2006) of C. gloeosporioides on mango.
| Fig. 4: | The effect of antagonistic bacteria against (a) Bipolaris sp. (b) Colletotrichum gloeosporioides (b) Botryosphaeria sp. and (d) Monilinia sp., under in vitro condition |
The alkaline pH environment for Bipolaris sp., tested in this recent experiment was supported by other earlier study. Bischoff and Garraway (1987) assumed that accumulation of ammonium and increasing pH on solid media could augment the production of NADP-glutamate dehydrogenase and NAD-glutamate dehydrogenase in liquid culture of Bipolaris maydis which might be associated with either the presence of mycelium or the presence of mycelium with conidia.
The optimum pH (pH 5.5) for C. gloeosporioides found in this study (Fig. 1b) was parallel with a number of earlier researches. Tandon and Chandra (1962) reported that pH range of 4.0-6.5 was optimum condition for growth and sporulation of C. gloeosporioides and then Maccheroni et al. (2004) studied that acidic pH (pH 5.0) was favorable condition for pectin secretion as the indication of polygalacturonase activity in pathogenic C. gloeosporioides.
The non significant inhibitions of given salinity levels against growth of tested fungi in this trial are in agreement with some results obtained by earlier studies. The in vitro assay conducted by Hopkins and McQuilken (2000) resulted in the fastest hyphal extension rates of Pestalotiopsis sydowiana, the causal agent of foliage, stem-base and roots diseases on hardy ornamental crops at commercial nurseries in the UK occurring on PDA osmotically adjusted with NaCl. In the field study, MacDonald (1982) observed that salinity stress treatments could enhance the development of root rot lesion on Chrysanthemum caused by Phytophthora cryptogea as high 70 and 88% of roots previously exposed to 0.1 and 0.2 M, respectively.
The findings of this current study, however, differed with the field investigation carried out by Elmer (2003), who studied the role of NaCl in suppression of crown and root rot disease on asparagus (Asparagus officinalis L.) caused by F. oxysporum or F. proliferatum. He discovered that the reduction in root lesions was significantly higher on roots which were directly exposed to NaCl (51% reduction) than on non-exposed roots (31% reduction).
This in vitro assay demonstrated that B. multivorans was the most efficacious biocontrol agent compared to others, excluding against Monilinia sp. Dikin et al. (2007) reported that the antimicrobial substances of this bacterium could restrict the growth of Schizophyllum commune, the causal pathogen of brown germ and seed rot disease on oil palm.
This current experiment discovered that B. cepacia had the maximum percent of inhibition (93.11%) at 2 DAI against Monilinia sp. Previously, Janisiewicz and Roitman (1988) discovered that B. cepacia could reduce in vitro growth and conidia germination of the stone fruit pathogen, M. fructicola by producing pyrrolnitrin. This recent study was not in agreement with the earlier experiment conducted by Bosch et al. (1992), who obtained the minimum inhibition (23%) of B. cepacia (syn. Pseudomonas cepacia) against M. fructicola causing brown rot on peach.
Against Bipolaris sp., this experiment revealed that B. cepacia could decrease its radial growth by 61.8% of optimum inhibition level for 8 days incubation. This was in range of inhibition level previously observed by (Jayaswal et al., 1993) on produced diffusible antifungal compounds against Helminthosporium maydis (syn. Bipolaris maydis) with around 58-75% for 3-5 days incubation but lower than against H. turcicum (syn. Bipolaris turcicum) with 76-83%..
On the other hand, the finding on B. cepacia against C. gloeosporiodes reported in this study showing 75.65% as optimum inhibition was higher than its inhibition on mycelial growth of the same pathogen isolated from anthracnose disease on papaya in post harvest storage (around 74.13%) (Rahman et al., 2007). In more recent study, Kadir et al. (2008) reported that higher dilution (1:8) of the antifungal substances in crude supernatant of B. cepacia was able to reduce both the mycelial growth and spore germination of C. gloeosporioides from similar isolate around 41 and 100%, respectively.
Differed with earlier study of B. cepacia effect on Diplodia maydis (syn. Botryosphaeria maydis) resulting in 42% of maximum inhibition level by the diffusible antifungal compounds for 5 days incubation (Jayaswal et al., 1993), this current study obtained higher inhibition level of B. cepacia on Botryosphaeria sp., namely 53.99% at 4 DAI. Jayaswal et al. (1993), however, showed about 75% optimum inhibition by the volatile compounds of this bacterium on D. maydis during 5 days incubation.
On other pathogenic fungi, Rahman et al. (2007) highlighted 68.45% of P. aeruginosa inhibition on the growth of C. gloeosporioides causing anthracnose disease on post harvest storage of papaya (Carica papaya L.) in Malaysia during in vitro screening on PDA medium. It was higher than that found on same fungi in this study, namely 55.41% of optimum inhibition at 4 DAI.
The absolute inhibitions were reached when the produced diffusible substances and bacterial suspension of P. aeruginosa were examined on mycelial growth of C. gloeosporioides isolate from papaya (Rahman et al., 2007). On the contrary, the lower effects were provided by its volatile substances on radial growth and spore germination, by its suspension (14.36 and 3.7%, respectively) and by its filter-sterilized culture filtrate on those two parameters (16.91 and 1.31%, respectively) of similar fungal isolate (Rahman et al., 2007).
CONCLUSION
In conclusion, the mycelial growth of tested fungi was highly affected by extreme condition of temperature (35°C) and pH (pH 4.0). Only concentration of 100 ppm could significantly inhibit the radial growth of certain given fungi; while among employed antagonistic bacteria, Burkholderia multivorans was the most effective in restricting the growth of the test fungi followed by B. cepacia and Pseudomonas aeruginosa in vitro. The proper combination of environmental modification may be useful for the growth of crop in the field as well as the storage life of the fruit at post harvest preservation.
ACKNOWLEDGMENTS
This study was conducted as the part of research project funded by the Fundamental Research Grant Scheme (FRGS) project No. (01-01-07-094FR) (2008/2009) under the Ministry of Higher Education, Malaysia. The authors also acknowledge Dr. Yahya Awang for suggestions in experimental design and model of statistical analysis.