Hardian Susilo Addy

Loss of Virulence of the Phytopathogen Ralstonia solanacearum Through Infection by φRSM Filamentous Phages

φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.

The Filamentous Phage ϕRSS1 Enhances Virulence of Phytopathogenic Ralstonia solanacearum on Tomato

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. ϕRSS1 is a filamentous phage that infects R. solanacearum strains. Upon infection, it alters the physiological state and the behavior of host cells. Here, we show that R. solanacearum infected by ϕRSS1 becomes more virulent on host plants. Some virulence and pathogenicity factors, such as extracellular polysaccharide (EPS) synthesis and twitching motility, increased in the bacterial host cells infected with ϕRSS1, resulting in early wilting. Tomato plants inoculated with ϕRSS1-infected bacteria wilted 2 to 3 days earlier than those inoculated with wild-type bacteria. Infection with ϕRSS1 induced early expression of phcA, the global virulence regulator. phcA expression was detected in ϕRSS1-infected cells at cell density as low as 10(4) CFU/ml. Filamentous phages are assembled on the host cell surface and many phage particles accumulate on the cell surface. These surface-associated phage particles (phage proteins) may change the cell surface nature (hydrophobicity) to give high local cell densities. ϕRSS1 infection also enhanced PilA and type IV pilin production, resulting in increased twitching motility.

Utilization of Filamentous Phage ϕRSM3 to Control Bacterial Wilt Caused by Ralstonia solanacearum

The wide host range of Ralstonia solanacearum, causal agent of bacterial wilt, and its ability to survive for long periods in the environment restrict the effectiveness of cultural and chemical control measures. The use of phages for disease control is a fast-expanding trend of plant protection with great potential to replace chemical measures. The filamentous phage ϕRSM3 that infects R. solanacearum strains and inactivates virulence on plants is a potential agent for controlling bacterial wilt in tomato. We demonstrated that inoculation of ϕRSM3-infected cells into tomato plants did not cause bacterial wilt. Instead, ϕRSM3-infected cells enhanced the expression of pathogenesis-related (PR) genes, including PR-1a, PR-2b, and PR7, in tomato plants. Moreover, pretreatment with ϕRSM-infected cells protect tomato plants from infection by virulent R. solanacearum strains. The effective dose of ϕRSM3-infected cells for disease prevention was determined to be approximately 105 CFU/ml. Because the ϕRSM3-infected cells can grow and continue to produce infectious phage particles under appropriate conditions, ϕRSM phages may serve as an efficient tool to control bacterial wilt in crops.

Detection of Bacterial Wilt Pathogen and Isolation of Its Bacteriophage from Banana in Lumajang Area, Indonesia

Bacterial wilt disease on banana is an important disease in Lumajang District and causes severe yield loss. Utilizing bacteriophage as natural enemy of pathogenic bacteria has been widely known as one of the control strategies. This research was aimed at determining the causing agent of bacterial wilt on banana isolated from Lumajang area, to obtain wide-host range bacteriophages against bacterial wilt pathogen and to know the basic characteristic of bacteriophages, particularly its nucleic acid type. Causative agent of bacterial wilt was isolated from symptomatic banana trees from seven districts in Lumajang area on determinative CPG plates followed by rapid detection by PCR technique using specific pair-primer. Bacteriophages were also isolated from soil of infected banana crop in Sukodono District. Morphological observation showed that all bacterial isolates have similar characteristic as common bacterial wilt pathogen, Ralstonia solanacearum. In addition, detection of FliC region in all isolates confirmed that all isolates were R. solanacearum according to the presence of 400 bp of FliC DNA fragment. Moreover, two bacteriophages were obtained from this experiment (RSSKD1 and RSSKD2), which were able to infect all nine R. solanacearum isolates. Nucleic acid analysis showed that the nucleic acid of bacteriophages was DNA (deoxyribonucleic acid).

Host range and molecular characterization of a lytic Pradovirus-like Ralstonia phage RsoP1IDN isolated from Indonesia

A lytic Ralstonia solanacearum-infecting phage designated Ralstonia phage RsoP1IDN was isolated from soil in Indonesia. The phage has a linear double-stranded DNA genome of 41,135 bp with 413-bp terminal repeats, and contains 41 annotated open reading frames. The phage is most closely related to Ralstonia phage RSB1, but different from RSB1 mainly in containing a putative HNH homing endonuclease and having a narrower host range. Our phylogenetic and genomic analyses revealed that both phages RsoP1IDN and RSB1 belong to the genus Pradovirus or a new genus, and not Phikmvvirus as previously reported for phage RSB1. RsoP1IDN is the first sequenced and characterized R. solanacearum-infecting phage isolated from Indonesia in the proposed species Ralstonia virus RsoP1IDN.

Antimicrobial Activity of Tithonia diversifolia , Elephantopus scaber , and Kigelia africana Against Plant Pathogens

Synthetic pesticides, one among agricultural inputs, have been used and applied to crop production, particularly during plant pathogen attacks. Although promisingly, possible effect that the application of pesticides on agro-ecosystem may have to be concerned to support health of food, consumers and to the environment. Alternatively, exploration of the potential plants that probably have natural antimicrobial compounds is important step to discover natural pesticide as component of plant disease management. Some of plants with low economical value such as Tithonia diversifolia, Elephantopus scaber, and Kigelia africana have been known to have antimicrobial substances and successfully demonstrated against food and human pathogens. These, bring us to study their potency in controlling several plant pathogens of important crops, either fungal such as Phytophthora nicotianae and Rhizoctonia solani, or bacterial pathogens such as Ralstonia solanacearum and Xanthomonas oryzae. Leave extracts of both, T. diversifolia and E. scaber, and fruit extract of K. africana were obtained and concentrated using methanol. Our results showed that all extracts contained flavonoid, tannin, and alkaloid but the amount of the content of each extract was different. Among extracts used in this study, fruit extract of K. africana was known to contain the highest flavonoid and tannin content of 21, 54 μg QE/ml and 28.95 μg GAE/ml, respectively, with low content on alkaloid (3.32 μg AE/ml) compared to other plant extracts. To test it potency as biopesticides, antimicrobial activity against fungal pathogens were evaluated using poisoned food technique method while antimicrobial activity against pathogenic bacteria were evaluated using spot test method. The result showed that extract from K. africana fruit was able to inhibit fungal pathogen R. solani, while extracts of E. scaber and T. diversifolia were have inhibition ability against P. nicotianae. In addition, the E. scaber extract was also able to inhibit bacteria R. solanacearum and X. oryzae. In average, 5 mg/ml of extracts were demonstrated to give the best performance in inhibit plant pathogens.

The involvement of the PilQ secretin of type IV pili in phage infection in Ralstonia solanacearum

PilQ is a member of the secretin family of outer membrane proteins and specifically involved in type IV secretion. Here we report the effects of pilQ mutation in Ralstonia solanacearum on the host physiology including susceptibility to several phage types (Inoviridae, Podoviridae and Myoviridae). With three lines of cells, namely wild type, ΔpilQ and pilQ-complemented cells, the cell surface proteins, twitching motility and sensitivity to phages were compared. SDS-PAGE analysis revealed that the major TFP pilin (PilA) was specifically lost in pilQ mutants and was recovered in the complemented cells. Drastically inactivated twitching motility in pilQ mutants was recovered to the wild type level in the complemented cells. Several phages of different types including those of Inoviridae, Podoviridae, and Myoviridae that infect wild type cells could not form plaques on pilQ mutants but showed infectivity to pilQ-complemented cells. These results indicate that PilQ function is generally required for phage infection in R. solanacearum.

Synergism of Lumbricus rubellus and Pseudomonas putida Pf-20 in Inducing Resistance to Cucumber Mosaic Virus

Both Lumbricus rubellus and Pseudomonas putida decompose soil organic matters. The population of P. putida Pf-20 increased if L. rubellus was introduced to the cucumber growth medium. The process of organic decomposition was much better if the medium was introduced with both L. rubellus and P. putida Pf-20, compared to the medium contained only either one of those organisms. The activity of L. rubellus may serve to provide nutrients for both the cucumber and P. putida. The role of P. putida to reduce disease severity was increased if L. rubellus was introduced to the growth medium. The synergism of these two organisms, reduced either the level of disease severity to CMV-48 and C/N ratio of medium, but increased the content of available phosphor and potassium. Key words: Lumbricus rubellus, Pseudomonas putida Pf-20, disease severity of CMV

Sinergisme Lumbricus rubellus dengan Pseudomonas putida Pf-20 dalam Menginduksi Ketahanan Mentimun terhadap Cucumber Mosaic Virus

Both Lumbricus rubellus and Pseudomonas putida decompose soil organic matters. The population of P. putida Pf-20 increased if L. rubellus was introduced to the cucumber growth medium. The process of organic decomposition was much better if the medium was introduced with both L. rubellus and P. putida Pf-20, compared to the medium contained only either one of those organisms. The activity of L. rubellus may serve to provide nutrients for both the cucumber and P. putida. The role of P. putida to reduce disease severity was increased if L. rubellus was introduced to the growth medium. The synergism of these two organisms, reduced either the level of disease severity to CMV-48 and C/N ratio of medium, but increased the content of available phosphor and potassium.