양송이배지로부터 분리한 인산가용화균 Burkholderia contaminans PSB-A와 Burkholderia ambifaria PSB-B의 단일접종 및 동시접종에 따른 인산가용화 능력의 시너지 효과 및 상추생육효과를 조사하였다. 인산캄슘이 함유된 NBRIP 액체배지에 분리균을 접종하고 해리된 가용인산 (soluble phosphorus)함량을 HPLC에 의해 분석하여 인산 가용화능을 측정한 결과, 배양 3일차에 동시접종구 166.3 μg mL -1 , B. contaminans 143.7 μg mL -1 , B. ambifaria 127.1 μg mL -1의 순으로 나타나 두 종의 인산가용화세균 간의 동시접종에 의한 시너지효과가 다소 관찰되었다. 배지 내 pH 와 잔류 glucose 함량 변화도 모든 접종구에서 배양 1 - 3일 후 pH 7.0에서 pH 4.0 수준으로 감소하였으며, glucose 함량은 초기 10 mg mL -1 에서 4.3 mg mL -1 수준으로 검출되어 인산가용화능과 거의 유사한 경향을 보였다. 배양여액의 유기산 분석결과 gluconic acid 약 12.3 mg mL -1 와 malic acid 약 7.9 mg mL -1 수준으로 가장 높게 검출되었으며, 결과적으로 두 세균이 분비하는 유기산이 배지의 pH를 감소시키고, 인산 가용화를 유도하는 주요 원인임을 확인할 수 있었다. 접종 4주 후 상추 생육차이를 조사한 결과, TCP를 첨가하지 않은 B. contaminans 접종구, B. ambifaria 접종구와 동시 접종구 에 비해 TCA 첨가한 모든 처리구에서 약 7-9% 수준의 생육증진효과가 보였으나, 단일접종과 동시접종구간에 유의수준의 차이를 확인할 수 없었다.

고추는 전 세계적으로 중요한 경제작물이다. 하지만, 매년 Colletotrichum species에 의해 발생하는 고추탄저병에 의해 상품성이 떨어지고 수량이 현저히 감소하여 많은 농가에 중대한 피해를 주는 주요 병해이기 때문에 방제대책이 절실히 요구되고 있다. 본 연구에서는 국내에 최근 발생한 C. scovillei에 대해 Burkholderia cepacia를 이용하여 길항 능력을 확인하였다. 16S rRNA와 recA 유전자 분석을 통해 분리된 균주가 B. cepacia임을 확인하였고, 식물 생장 촉진 물질인 siderophore와 항진균 가수분해 효소인 protease, cellulase 생산을 기내 실험을 통해 확인하였다. 또한, C. scovillei에 대한 KF1의 균사 생장 억제율은 66.0%로 나타났으며, 1×105, 106, 107, 108 cfu/mL의 길항 미생물 처리 농도에 따른 포자 발아율은 각각 7.8%, 4.3%, 1.2%, 0%로 확인되어 분리된 길항 미생물은 효과적으로 병원균의 균사 생장 및 포자 발아를 억제하였다. 따라서 본 연구는 길항 미생물 B. cepacia KF1의 고추탄저병균에 대한 생물학적 방제제 자원으로서의 활용가능성을 제시하였다.

In general, hemipteran insects transmit either obligate or facultative symbionts to their offspring using various transmission mechanisms. Riptortus pedestris (Hemiptera: Alydidae) also have facultative symbiosis with Burkholderia sp. which is acquired from the soil every generation especially during 2nd instar nymphal period. This environmental symbiont transmission makes R. pedestris face a great risk of missing their symbionts, however, previous studies showed that they have intimate associations with their symbionts. Therefore, we suspected that R. pedestris 2nd instar nymphs may have a preference toward Burkholderia sp. to achieve an efficient symbiont acquisition during the limited time of window. In order to identify the preference of 2nd instar nymph, we first conducted dual-choice experiments varying with the number of 2nd instar nymphs (a nymph and 100 nymphs) in plastic cages. Furthermore, Y-tube olfactometer experiments varying with the presence of soil as medium of Burkholderia sp. were performed to determine if the insects respond to bacterial volatile induced from the symbiont. Based on our results, we observed that R. pedestris 2nd instar nymphs may not show any specific behavioral preference or response to chemical volatile from Burkholderia sp. in laboratory conditions (P >0.24). Therefore, we will further conduct a dual-choice experiment in a semi-field condition to investigate if such patterns can be detected in a larger arena simulating a more natural environment.

양송이 수확후 배지에서 분리한 Pantoea rodasill LH-1와 Burkholderia stabilis HS-7간의 단일 접종 및 동시접종에 따른 인산가용화능과 상추생육효과를 조사하였다. NBRIP액체 배지에 분리균을 접종하고 배지에 함유된 인산칼슘으로부터 해리된 가용인산(soluble phosphorus)함량을 측정한 결과, P. rodasill LH-1는 736.59 μg mL-1, B. stabilis HS-7은 743.90 μg mL-1, 그리고 B. stabilis HS-7와 P. rodasill LH-1을 동시 접종한 배지는 783.41 μg mL-1로 가장 높았지만 유의수준의시너지 효과는 나타나지 않았다. 배지 내pH는 초기 pH 7.0에서 배양 3일 후 모든 접종구에서 pH 4.3수준으로 감소하였으며, 또한 배지 내잔류 당 감소율은 P. rodasill LH-1 31.7%, B. stabilis HS-7은 53.7%, 동시 접종한 배지에서는 87.1%로 가장 높은 감소율을 나타냈다. 분리균에 의해 생성한 배지 내유기산을 분석한 결과, gluconic acid, lactic acid, malic acid, acetic acid, citric acid 이 검출되었으며, 이 중 citric acid가 주요 유기산으로 동시 접종한 배지에서 0.96 mg mL-1로 가장 높은 함량을 나타내었다. 상추 재배 실험결과 동시 접종구가 대조구에 비해 잎의 길이는 7.7%, 뿌리길이는 15.5% 생육증진효과를 보였다. 이 결과들을 바탕으로 B. stabilis HS-7 와 Pantoea rodasill LH-1이 동시 접종에 의해 유의수준의 인산가용화 시너지 효과는 관찰되지 않았지만, 무 접종구에 비하여 배지 내 인산가용화능과 작물의 생장이 크게 촉진되는 결과를 보임으로서 인산가용화균을 이용한 미생물 비료의 공급으로 염류집적토양의 인산가용화능과 작물의 생육을 향상시킬 수 있다고 생각된다.

This study investigated the synergistic effect of single inoculation and co-inoculation of phosphate-solubilizing bacteria (PSB) Burkholderia metallica JH-7 and Burkholderia contaminans JH-15. Phosphate-solubilizing abilities of these strains were assessed by measuring phosphorus content in culture media that were singly inoculated or co-inoculated with these strains for 7 days. B. metallica JH-7 was found to release the highest content of soluble phosphorus (140.80 μg mL-1) into the medium, followed by single inoculation of B. contaminans JH-15 (135.95 μg mL-1) and co-inoculation of two strains (134.84 μg mL-1). The highest pH reduction, organic acid production, and glucose consumption were observed in the medium inoculated with B. metallica JH-7 alone compared with that in the medium co-inoculated with both the strains. Results of a plant growth promotion bioassay showed 17.4% and 7.48% higher leaf and root growth, respectively, in romaine lettuce inoculated with B. metallica JH-7 alone than in romaine lettuce inoculated with a control strain. However, no significant difference was observed between single inoculation and co-inoculation of these strains with respect to phosphorus release and plant growth. Although the results of the present study did not show the synergistic effect of phosphate solubilization by the PSB strains examined, these results indicate that treatment with PSB exerts a beneficial effect on crop growth.

Vertical transmission of symbiont is known as the most effective way to deliver their offspring. However, it has beenreported that the bean bug, Riptortus pedestris, acquires its gut symbiont, Burkholderia sp., from its environment (e.g.soil) during the 2nd instar nymphal stage. Nevertheless, it is not unknown how they locate their symbiont. For this reason,dual-choice experiments were conducted in both solitary (a nymph) and group conditions (100 nymphs) to investigatewhether or not R. pedestris has preference on Burkholderia sp. treated or untreated solution in laboratory conditions. Inthese experiments, there was no significant difference in the preference between the two treatments. We are testing inthe Y-tube olfactometer system to evaluate the response of R. pedestris on possible chemical cues from Burkholderia sp..

The bean bug, Riptortus pedestris, is a pest of leguminous crops. Recent studies have revealed an interesting relationship between R. pedestris and a gut symbiont Burkholderia sp.. R. pedestris mainly acquires Burkholderia sp. during 2nd instar period, which leads to enhancement of growth and reproductive rate of the host. Nevertheless, how R. pedestris acquires its symbiont is not yet clearly described. Therefore, we investigated whether 2nd instar nymphs have preference on Burkholderia sp. compared to untreated control in laboratory settings. In the choice test, no significant difference was found in the preference by R. pedestris between the two treatments. To further investigate single individual’s behaviors (its first choice, approaching frequency to each treatment, and residence time on the treatment). We video-recorded single individuals in the choice setting for 12 hours. Again, there was no significant difference in any of the behavioral traits recorded. Further choice tests will be conducted in the Y-tube olfactometer systems and semi-field conditions.

In order to establish symbiotic host-bacterial relationships, symbionts in insects evolved a mechanism to overcome host immune responses. Here we provide the resistance of symbiotic bacteria on the insect immune system. As a result, through the transposon mutagenesis, we found a salivary gland (SG) susceptible mutant. The disrupted gene was identified as nlpB involved in lipoprotein synthesis. The nlpB, bla double deletion mutant was sensitive to SG like nlpB-Tn5 inserted mutant. This mutant increases outer membrane permeability. It provides an explanation for SG susceptibility, because the antimicrobial peptide in SG would be able to translocate across the outer membrane more easily than in the wild type. These results indicate that nlpB and bla are likely to be important factors in terms of determining resistance against SG of Riptortus that is connected with the successful colonization of the Riptortus midgut.

Many insects possess symbiotic microorganisms in their bodies, wherein host-symbiont intimate interactions occur. Despite recent advances in omics technology, the molecular bases of the symbiotic associations remain unclear. The bean bug Riptortus pedestris harbors an environmentally acquired Burkholderia symbiont in their midgut crypts. Unlike other insect symbionts, the Burkholderia symbiont is easily culturable and genetically manipulatable outside the host insect. In conjunction with the experimental advantages of the bean bug, the Riptortus–Burkholderia association is an ideal model system for approaching the molecular bases underpinning insect-microbe symbioses. In this presentation, the current knowledge of this model system is summarized.

Recent studies suggested that gut symbionts modulate insect development and reproduction. However, how gut symbionts modulate host physiologies and what types of molecules are involved in these changes are still unclear. When we analyzed hemolymph proteins and transcriptional levels of host insects, hexamerin-α (Hex-α), hexamerin-β (Hex-β) and vitellogenin-1 (Vg-1) were highly expressed in symbiotic insects (Sym) compared to aposymbiotic insects (Apo). Depletion of Hex-β by RNA interference in 2nd Sym-nymphs delayed adult emergence, whereas Hex-α and Vg-1 RNA interference in 5th nymphs decreased reproduction of female insects and caused loss of color of laid eggs. Also, the levels of JHSBIII of Riptortus host were 3-fold higher in the Sym-female insects compared to the Apo-insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production through regulating the expression of three host storage proteins by controlling of brain hormone.

Five phaP family genes and one phaR gene have been identified in the genome of Burkholderia gut symbiont. PhaP proteins function as surface proteins of polyhydroxyalkanoate (PHA) granules, and PhaR protein acts as a negative regulator of PhaP biosynthesis. To address the biological roles of four phaP family genes (phaP1, phaP2, phaP3, and phaP4) and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second instar nymph. The ΔphaR mutant decreased the colonization ability in the host midgut compared to wild-type Burkholderia cells and negatively affected the host insect’s fitness compared with wild-type infected host. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects’ midgut