THE EFFECT OF FLUORESCENT PSEUDOMONADS APPLICATION ON THE RESISTANCE OF CHILI PLANTS AGAINST THE ATTACK OF RALSTONIA S OLANACEARUM AND FUSARIUM OXYSPORUM IN THE FIELD

Chili (Capsicum annuum L.) is one of the vegetable commodities with high economic value. Until now, the necessity rate of chili is still high but the production is still low. One of the obstacles, which highly influence chili production, is wilt disease due to Ralstonia solanacearum and Fusarium oxysporum. This study aims at identifying the best fluorescent pseudomonads isolate in suppressing the development of the primary wilt disease due to Ralstonia solanacearum and F. oxysprorum in the field. This study employs Completely Randomized Design that comprises 3 treatments. The three treatments are the type of fluorescent pseudomonads which is Pf-122 isolate, Pf-160 isolate, and Pf-B isolate. The observation parameter on suppressing the disease development is perceived from incubation period of the initial appearance of symptoms, disease index and the change of stem tissue color. The result of the study shows that the longest incubation period is treatment with fluorescent pseudomonads of Pf-122 isolate and Pf-160 isolate. The chili is treated with fluorescent pseudomonads of Pf-122 isolate and Pf-160 isolate, the initial appearance of symptoms at the 8th day after inoculation. In the treatment with fluorescent pseudomonads of Pf-122 isolate and Pf-160 isolate compared to control, is able to delay the appearance of symptom for 3 days. The disease development starts from 10th to 30th day, the treatment with Pf-122 isolate shows the slowest development compared to other treatment and control. In the last observation, the disease index with treatment of Pf-122 isolate is the lowest which is 37%, then it is followed by Pf-B and Pf-160 which are 39% and 40% respectively, whereas in the control it reaches 67%. For the color change on stem tissue, there is no real difference of the three treatments of fluorescent pseudomonads application but it differs and smaller from the control.


INTRODUCTION
Chili productivity in Indonesia is currently low.The factors causing low production are the use of less qualified seeds, non-efficient cultivation technique and the chili cultivar cultivation which is not resistant to pests and diseases (Soelaiman & Ernawati, 2013).The primary barrier which highly influences the chili production is the main disease, wilt disease Ralstonia solanacearum and Fusarium oxysporum.Disadvantages from wilt disease of R. solanacearum are quite great (Elphistone, 2005).In Indonesia, this disease causes a very big disadvantage and the disease intensity can reach 75% (Fortman & Martin, 1998).
The disadvantage due to this disease is increasingly high for in the field it commonly attacks chili joint together and synergic.Numerous attempts to overcome the main disease of being wilt disease on chili have been sought to, but an effective and efficient controlling method has not been discovered.It is because both pathogens can withstand in the soil and both of them synergize to attack chili.Therefore, an attempt to overcome the pathogen attack and to improve the chili resistance is necessary.
Biological control can improve the result by suppressing pathogen inoculum, protecting the plant against infection, and improving the plant resistance from pathogen.Microorganism will block pathogen at the root.Useful biological control with microorganism can induce root growth will be increasingly performed (Podile & Kishore, 2007).
Certain strain from fluorescent pseudomonads shows its ability in suppressing the development of several plant diseases due to pathogens and inducing plant growth (Raaijmakers et al., 1995).Primary mechanism of suppressing disease by biological agents are including antagonism and or inducing plant resistance (Hyakumachi, 2013).Bacteria are able to induce plant systemic resistance by producing phytohormone, solubilizing inorganiz phosphate, improving fastening of Fe and siderophore (Podile & Kishore, 2007).As suggested by Pinton et al. (2001), rhizobacteria is also able to induce plant growth by producing growth regulator such as auxin derivatives.Most of Pseudomonas, Bacillus, Azotobacter rhizobacteria produce growth inducing compound such as Indol Acetic Acid (IAA), gibberellins, and other substances such as cytokinin (Brimecombe et al., 2001).Antibiotics produced by fluorescent pseudomonads contributing in biological control are phenazines, 2.4diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide (Haas and Keel, 2003).Antibiosis is activity of response generally from several biological agents such as Pseudomonas spp, Bacillus spp, Streptomyces spp, and Trichoderma spp.(Alabouvette et al., 2006).Wuryandari et al., (2007) showed that fluorescent Pseudomonad of Pf-122 isolate can inhibit bacterial wilt disease caused by Ralstonia solanacearum.In the last observation (30 days) the disease index is only 49.9% at greenhouse scale.To determine the ability of fluorescent Pseudomonad to improve the plant resistance toward 2 pathogens working in synergy in the field (R. solanacearum and F. oxysporum), research needs to be done by using fluorescent pseudomonad to induce chili plant resistance toward the primary wilt disease due to R. solanacearum and F. oxysporum in the field.

METHODOLOGY
The study is conducted in land commonly used for Solanaceae plant cultivation and frequently exhibited the symptom of wilt disease, which is in Lebo village, Sukodono, Sidoarjo, East Java, Indonesia.The study employs Completely Randomized Design comprising 3 treatments.The three treatments are the types of fluorescent pseudomonads isolate which is Pf-122 isolate, Pf-160 isolate, and Pf-B isolate.Each treatment has three repetitions.Control treatment as the comparator is chili without treatment with fluorescent pseudomonads The root of chili seeds aged 30 days are immersed into fluorescent pseudomonads suspension for 30 minutes, then it is planted.Negative comparator consists of chili seed immersed into sterile water for 30 minutes and is planted in the soil.The concentration of fluorescents pseudomonads used is 10 10 cfu/ml.Each treatment is repeated three times.To identify plant resistance toward wilt disease due to R. solanacearum and F. oxysporum, observation parameter includes incubation period, disease index, and the color change of stem tissue.
Incubation period is observed from pathogen inoculation until the appearance of wilt disease symptom on chili.The disease index is observed in the development of wilt disease symptom 5 days / plant until 30th day after inoculation.For the color change of stem tissue is observed at the 30th day, by splitting length wise the stem and measuring the length of color change in vascular tissue.
The weight of attack is estimated according to the following scale: 0 = no symptom, 1 = 1 to 10% wilt leaves, 2 = 11 to 30% wilt leaves, 3 = 31 to 60% wilt leaves, 4 = 61 to 99 % wilt leaves, and 5 = 100% wilt leaves The magnitude of disease index is estimated by using a formula (Arwiyanto, 1995): The formula description is as follows; I = disease index, nk = the number of plant having disease symptom with scale k (0, 1, 2, 3, 4, 5), N = total number of inoculated plant, and Z = highest category of attack.

RESULTS AND DISCUSSION a. Incubation period
If it is seen from the results of observation on incubation period of wilt disease, the three fluorescent pseudomonads show different result.Incubation period of wilt disease is fastest in the treatment of Pf-B isolate then in Pf-122 isolate and Pf-160 isolate which are 7 days and 8 days.Control in incubation period is 5 days.If compared to control then the treatment with Pf-122 isolate and Pf-160 isolate it is able to delay the emergence of the symptom for 3 days, whereas with Pf-B isolate it is only 2 days (Figure 1).

Figure-1. Incubation period of wilt disease symptoms on chilli
Short delay of incubation period of wilt disease by fluorescent pseudomonads rhizobacteria, it is possible due to lack of fluorescent pseudomonads population, which may inhibit pathogen attack.The dose of pseudomonad fluorescents rhizobacteria used to plant submergence is probably still less high, so that it is not quite able maximally in inhibiting pathogen to infect chili.The result of a study conducted Wuryandari et al. 2012, that chili treated with fluorescent pseudomonads of Pf-160 isolate, Pf-B isolate and Pf-122 isolate and is inoculated with F. oxysporum, able to delay the disease symptom longer which is 13 days, 12 days, and 6 days.The difference of incubation period delay is possible because beside pathogen which has to be inhibited is not only one, but also because different application scale.Previous research application is in greenhouse, whereas this study is conducted in the field.Field condition with more complex microorganism so that it can influence the ability of pseudomonad fluorescents in expressing the ability in producing inhibiting compound to resist pathogen and the ability in producing growth hormone for chili.Biological control of pathogen by rhizobacteria can be based on bacteria metabolite such as siderophores, antibiotics, and hydrogen cyanide.Rhizobacteria can also induce the plant growth by producing growth regulator such as auxin derivatives (Pinton et al, 2001).

b. Disease Index
The development of disease stating from inoculation until 30th day of observation after inoculation can be seen in Figure 2. In the last observation, the treatment with Pf-122 isolate shows the lowest disease index which is 37% then it is followed by Pf-8 isolate and Pf-160 isolate which are 39% and 41%, whereas in the control it reaches 67%.If it is compared to control, then the submergence by Pf-122 isolate is able to suppress the disease development by 30%, whereas it is with Pf-B and Pf-160 isolates only able to suppress the disease development by 28% and 18% (Table 1) The result shows that the application of fluorescent pseudomonads can suppress the wilt disease by Fusarium and Ralstonia solanacearum.Fluorescent pseudomonads of Pf-122 isolate is the best in suppressing the wilt disease.It is possible because fluorescent pseudomonads is able to produce a compound which can inhibit pathogens of Fusarium and Ralstonia solanacearum and can induce chili growth so that it can be resistant to the pathogen attack.Primary mechanism in suppressing disease by biological agents includes antagonism and or inducing plant resistance (Hyakumachi, 2013).
The application of fluorescent pseudomonads in chili rooting, it probably causes fluorescent pseudomonads colonize root area so that it inhibits the pathogen population causing wilt disease.Mechanism of biological control of pathogens by rhizobacteria may be in form of bacteria metabolite production.Bacteria of Pseudomonas genus also produces antimicrobial compound such as phenazine, pyrrolnitin, pyluteorin, phloroglucinols, cyclic lipopeptides, and hydrogen cyanide (Haas & Defago, 2005).Application of pseudomonad fluorescens also allows the plant growth to be induced so that it can be more resistant to pathogen attack of Fusarium and Ralstonia solanacearum.As suggested by Pinton et al., (2001), rhizobacteria is also able to induce plant growth by producing growth regulator such as auxin derivatives.Most of Pseudomonas, Bacillus, Azotobacter rhizobacteria produce growth inducing compound such as Indol Acetic Acid (IAA), gibberellins, and other substances such as cytokinin (Brimecombe et al., 2001).The sufficient existence of fluorescent pseudomonads in rooting area is allegedly able to dissolve phosphate plays a role in repairing the plant.In accordance to Beattie (2007), one way to repair phosphorus deficiency on plant is by soil inoculation with phosphate solubilizing microorganism.Several saprophytic bacteria playing role as biological agents of wilt disease control is fluorescent pseudomonads bacteria on tomato, potato, and eggplant, and on chili (Saddler, 2005).Bacteria are able to induce plant systemic resistance by producing phytohormone, solubilizing inorganiz phosphate, improving fastening of Fe and siderophore (Podile & Kishore, 2007;Haas & Defago, 2005).

c. Color change on stem tissue
Based on the observation result on symptom of color change in stem vascular tissue, it is seen that the average symptom of color change in stem with the treatment using fluorescent pseudomonads, is smaller than control.Application using Pf-160 isolate shows the smallest change of color then followed by Pf-122 and Pf-B isolates (Figure 3).It is probably that fluorescent pseudomonads rhizobacteria applied is able to colonize in chili root so that it can inhibit microorganism in soil including pathogen causing wilt disease of R. solanacearum and Fusarium sp.The ability of pseudomonad fluorescents rhizobacteria of Pf-160 isolate in inhibiting disease is seemingly better compared to other fluorescent pseudomonads rhizobacteria.It is seen from the slow or small vascular tissue in the stem.Although fluorescent pseudomonads of Pf-160 isolate is better in inhibiting the damage on vascular tissue, it is apparently not different from other isolate fluorescent pseudomonads in arising symptom of wilt disease on plant.According to Baharuddin et al (2005), the result of his study explains that Pseudomonas spp.fluorescent group in plant rooting is able to colonize well so that it can suppress the attack by R. solanacearum.The application of fluorescent pseudomonads expected to be able to inhibit pathogens causing wilt disease which is R. solanacearum and Fusarium sp.living in rooting area before the pathogens entering the plant root tissue.

CONCLUSION
Application with pseudomonad fluorescent of Pf-122 isolate compared to control is able to postpone the emergence of the symptom for 3 days.However, if it is seen from the disease index, compared to other isolate and control, Pf-122 isolate shows the highest inhibiting ability.

ACKNOWLEDGEMENT
The

Figure- 2 .
Figure-2.The development of wilt disease on chili after treated with fluorescent pseudomonads rhizobacteriaThe result of disease index from the appearance of wilt disease symptom to the 30th day of observation, it is seen that the development of wilt disease on plant with fluorescent pseudomonads from the three isolates is slower

Figure- 3 .
Figure-3.Influence of fluorescent pseudomonads treatment on symptom length of color on stem

Table - 1
. The index of wilt disease on chili with treatment using fluorescent pseudomonads rhizobacteria at 30 th day author would like to thank Director of Research and Public Service, Directorate-Generals of Higher Education, Ministry of Education and Culture of