Ⅸ . 식물병의 진단 1. 병징과 표징 2. 병의 진단법

1. 병징과 표징 : symptom(병징) - the external and internal reactions or alterations of a plant as a result of a disease. - 식물체가 어떤 원인에 의하여 그 식물체의 세포․조직 혹은 기관에 이상이 생 겨서 외부 형태에 어떤 변화가 나타났을 때 : sign(표징) - the pathogen or its part or products (include mycelium, sclerotinia, sporophores, fruiting bodies, and spores) seen on a host plant. - 병원체가 병든 식물의 표면에 나타나서 눈으로 가려낼 수 있을 때 : 병징과 표징은 병의 종류에 따라 어느 정도의 특징이 있으므로, 이들을 잘 안다 는 것은 진단을 위해 중요하다.

(1) 병징 (a) 전신적 병징 ① 시들음병(萎凋病; 위조병; wilt disease)    - 토마토시들음병, 각종 모잘록병(damping-off)   ② 오갈병(萎縮病; 위축병; dwarf disease)    - 벼오갈병, 보리오갈병, 뽕나무오갈병   ③ 황화병(黃化病; chlorosis)    - 과꽃위황병, 복숭아나무황화병    * 오갈 현상이 겹쳐서 나타날 경우는 위황병(萎黃病, yellow)

(b) 부분적 병징   ① 혹이 만들어지는 병(excrescent disease) - 각종 식물의 뿌리혹병(根頭癌腫病, 근두암종병), 감자 암종병,  배추 무사마귀병(根瘤病, 근유병)   ② 빗자루병(天狗巢病; 천구소병; witches' broom)    - 대추나무 빗자루병, 벚나무 빗자루병, 오동나무 빗자루병,   ③ 가지마름병(枝枯病; 지고병; dieback)    - 감나무 가지마름병   ④ 줄기마름병(胴枯病; 동고병; canker)    - 밤나무줄기 마름병, 배나무 줄기마름병, 소나무류 줄기마름병(pitch canker) ⑤ 불마름병(燒病; 소병; blighting disease)    - 감자역병(疫病), 벼 잎집얼룩병, 배나무 화상병

  ⑥ 미이라병(mummification)    - 콩미이라병, 포도나무꼭지바름병, 사과나무검은빛썩음병   ⑦ 썩음병(腐敗病; 부패병; rotting disease)    - 벼모썩음병, 벼좀공균핵병, 벼잎집썩음병, 채소류무름병   ⑧ 잎말림병(葉捲病; 엽권병; curling disease)    - 감자잎말림병, 복숭아나무잎오갈병   ⑨ 대화병(帶化病; fasciation)    - 일종의 생리병

(c) 국부적 병징  ① 더뎅이병(瘡痂病; 창가병; scab disease) - 감자 더뎅이병, 감귤 더뎅이병, 콩더뎅이병   ② 궤양병(潰瘍病; canker disease)  - 감귤궤양병   ③ 구멍병(穿孔病; 천공병; shot-hole disease)  - 복숭아나무 세균성구멍병   ④ 점무늬병(斑點病; 반점병; spotting disease) – 벼 깨씨무늬병, 사탕무우 갈색무늬병, 감나무 둥근무늬낙엽병, 배나무 검은무늬병, 배추 흰무늬병   ⑤ 오반병(汚斑病; blotch) – 보리 구름무늬병, 사과나무 갈색무늬병   ⑥ 모자이크병(mosaic disease) – 담배 모자이크병, 오이 모자이크병   ⑦ 줄무늬병(條斑病; 조반병; strip disease) – 보리 줄무늬병, 밀 누른줄무늬병

* Symptoms caused by fungi on plants : In general, fungi cause local or general necrosis or killing of plant tissues and they often result in reduced growth (stunting) of plant organs or entire plants. : the most necrotic symptoms are as follows : - Leaf spots (잎점무늬) → Localized lesions on host leaves consisting of dead and collapsed cell. - Blight (마름증상) → General and extremely rapid browning and death of leaves, branches, twigs, and floral organs. - Canker (궤양) → Localized necrotic lesions on a stem, often sunken, of a plant. - Dieback (가지마름) → Extensive necrosis of twigs beginning at their trips and advancing toward their bases - Root rot (뿌리썩음) → Disintegration or decay of part or all of the root system of a plant.

- Damping-off (모잘록)     → Rapid death and collapse of very young seedlings   - Basal stem rot (기부줄기썩음)     → Disintegration of the lower part of the stem - Soft rots and dry rots (물렁썩이와 마른썩이)     → Maceration and disintegration of fruits, roots, bulbs, and fleshy leaves.   - Anthracnose (탄저)     → Necrotic and sunken ulcer-like lesion on the stem, leaf, fruit, or flower of the host plant caused by a certain group of fungi.    - Scab (더뎅이)    → Localized lesions on host fruit, leaves, tubers, etc., usually slightly raised or sunken and cracked, giving a scabby appearance.   - Decline (쇠퇴)    → Plants growing poorly; leaves small, brittle, yellowish, or red; some defoliation and dieback present.

: Symptoms associated with excessive enlargement or growth and distortion of plant parts include the following. - Clubroot (무사마귀, 근류병) → Enlarged roots appearing like spindles or club . - Galls (혹) → Enlarged portions of plant organs (stems, leaves, blossoms, roots). - Warts (사마귀) → Wart-like protuberances on tubers and stems. - Witches'-brooms (빗자루병) → Profuse, upward branching of twigs. - Leaf curls (잎오갈) → Distortion, thickening, and curling of leaves.

: In addition to the above.   - Wilt (시들음)    → Generalized loss of turgidity and drooping of leaves or shoots.   - Rust (녹병)    → Many small lesions on leaves or stems, usually of a rusty color.   - Smut (깜부기)    → Seed or a gall filled with the mycelium or black spores of the smut fungi.   - Mildew (노균병 downy- , 흰가루병 powdery-)    → Areas on leaves, stems, blossoms, and fruits, covered with whitish mycelium and the  fructifications of the fungus.

(2) 표징 (a) 가루(紛) - 흰가루병, 녹병, 흰녹가루병, 깜부기병, 떡병(진달래나무) 등 (b) 곰팡이    - 흰가루병, 녹병, 흰녹가루병, 깜부기병, 떡병(진달래나무) 등   (b) 곰팡이   - 솜털 모양(벼모썩음병, 가지솜털역병, 고구마무름병, 잔디면부병), 깃털모양 (과수류 날개무늬병), 잔털 모양(감자 겹둥근무늬병, 수박 덩굴쪼김병, 강낭 콩 모무늬병), 서릿발 모양(오이류 노균병, 감자 역병, 배나무 검은별무늬병)   (c) 흑색소립점(黑色小粒點)    - 병자각(사과나무부란병, 배나무줄기마름병), 자낭각(보리 붉은곰팡이병, 배 나무 뒷면흰가루병), 포자퇴(밀 줄기녹병), 자좌(밤나무 줄기마름병, 사과나 무 부란병)

 (d) 균핵(菌核; sclerotinia)   - 벼․채소․과수의 균핵병  (e) 돌기(突起)   - 배나무 붉은별무늬병(녹포자기), 고구마 검은무늬병(자낭각 돌기)  (f) 버섯   - 과수․수목 등의 뿌리썩음병, 채소류의 균핵병(자낭반)  (g) 끈끈한 물질(粘質物)   - 포자누출(胞子漏出; spore-ooze), 세균누출(細菌漏出; bacterial-ooze)  (h) 냄새   - 밀 비린깜부기병(비린내), 고구마 검은무늬병(쓴맛), 감자 무름병(나쁜냄새), 사과나무 부란병(알콜냄새)

2. 병의 진단법(診斷法) : 진단(diagnosis)이란? - 병든 식물체를 정밀하게 검사하여 비슷한 병과 구별하고, 바른 병 이름을 정하는 것 cf. 진단학(diagnostics) * As defined in Webster's Unabridged Dictionary, diagnosis is " the art or act of recognizing the presence of disease from its symptoms, and deciding as to its character; also the conclusion arrived at through critical perception or scrutiny; hence keen understanding of appearance

: 식물의 병 진단은 병 발생을 막는 데 기초가 되며, 올바른 진단을 할 때에 비로소 알맞은 방제법을 찾을 수 있고, 방제 효과도 기대할 수 있다. : 식물병의 진단에는 적은 수의 병든 식물체뿐만 아니라, 그 부근에 있는 다른 종류의 식물도 포함하여 피해의 발생 상황과 발생의 추이, 환경조건 등을 조사할 필요가 있다.

- 전염성병 → 같은 종류의 식물이라도 병든 개체와 건전한 개체가 섞여 있으며, 병든 식 물체 사이 혹은 같은 개체의 부분에 따라서도 병 정도의 차이가 다르다. → 병의 발생이나 퍼져 가는 정도가 주위의 환경조건이나 취급하는 방법에 따라 달라진다. ⇒ 발병상황, 발병지의 환경조건, 발병시기, 발생포장의 종류 등을 조사. ⇒ 발병개체(식물)의 屬, 種, 품종, 계통, 건강상태, 나이, 재배환경, 현재까지 의 경과 등을 조사. - 비전염성병 → 대부분의 식물체에서 똑같은 병징이 나타나는 경우가 많고, 다른 종류의 식물에도 비슷한 병징이 나타나는 일이 있다.

(1) 눈에 의한 진단  - 도열병에 걸린 벼 잎의 병 무늬는 색깔과 모양에 의해서 격발형, 만성형, 갈 점형 및 백점형으로 나눌 수 있다(환경조건과 품종의 차이).   - 표징은 절대적이고, 그 빛깔, 모양 크기 등이 대체로 일정하기 때문에 진단 에 결정적인 역할을 한다(자주날개무늬병, 흰날개무늬병, 그을음병, 맥각병, 흰비단병, 균핵병, 노균병, 잿빛곰팡이병, 흰가루병, 녹병, 흰녹가루병, 깜부기 병, 뿌리혹병, 빗자루병 등).

(2) 해부적 진단 - 병든 부분의 조직을 해부하여 조직속의 이상현상이나 병원체의 존재를 밝히는 방법   - 병든 부분의 조직을 해부하여 조직속의 이상현상이나 병원체의 존재를 밝히는 방법    → 감자잎말림병[줄기관다발의 괴사현상; 플로로그루신(phloroglucin)황산법 또는 나디 반응(nadi-reaction)을 이용하여 괴사부 발색)    → 바이러스병(세포내에 X-body라는 물질유․무 조사)    → 벼 좀공균핵병(볏짚의 땅 가장자리 부분을 쪼개면 그 속에 검고 작은 균핵이 밀생)    → 세균성인 벼 흰잎마름병이나 가지과식물의 풋마름병(잎 또는 줄기를 잘라 시험관의 물속에 넣으면 절단부에서 병원세균이 흰 연기모양으로 쏟아져 나옴)    → 진균성인 토마토 시들음병과 오이류 덩굴쪼김병은 뿌리나 줄기를 쪼개보면 물관부가 갈변)

(3) 병원적 진단   - 병원체를 현미경 또는 전자현미경으로 관찰하거나, 병원체를 분리, 배양, 접 종해서 병원성을 확인하는 방법으로 진단하는 것.    → Koch's postulates (4) 이화학적 진단   - 병든 식물의 이화학적 변화를 조사하여 병의 종류를 진단    → 감자둘레썩음병(씨감자를 쪼개서 자외선을 쪼이면 관다발부가 둥글게 형 과빛을 나타냄)   - 식물의 체질을 조사하여 발생예찰 등에 이용    → 벼도열병(잎의 질소함량, 가용태 질소함량, 규산함량과 가용태 질소함량 의 비, phenol 성분함량과 가용태 질소함량의 비, 혹은 잎집의 전분 축적량 을 옥도, 옥도칼륨액으로 염색)

(5) 혈청학적 진단 (a) 슬라이드법(slide method)   (b) 한천겔확산법(agar gel diffusion method)   (c) 형광항체법(fluoresvent antibody method)   (d) 적혈구응집반응(hemagglutination reaction test)   (e) 라텍스 응집반응(latex agglutination reaction)   (f) 효소결합항체법(ELISA test; enzyme linked immuno sorbent assay)

(6) 생물학적 진단 (a) 지표식물에 의한 진단 (b) 괴경 지표법에 의한 진단 (c) 충체내 주사법에 의한 진단   (d) 파지(phage)에 의한 진단   (e) 병든 식물의 즙액접종법   (f) 토양검진    ① 희석평판법    ② 유리종분산법(bell jar dust method)    ③ 포착법(捕捉法)    ④ 잔사법(殘渣法)    ⑤ 부상법(浮上法)    ⑥ 유식물검정법(幼植物檢定法)

Agglutination agglutinates e.g., Widal test visible clumps or aggregates of cells or particles e.g., Widal test diagnostic for typhoid fever e.g., latex agglutination tests pregnancy test Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Viral hemagglutination Figure 33.9a (top): Certain viruses can bind to red blood cells causing hemagglutination. Figure 33.9b (top): Diagnostic tests can be developed that are based on hemagglutination. In this case, mixing serum containing specific antibodies to the virus with red blood cells, neutralizes the virus and inhibits hemagglutination (a positive test). Figure 35.11 Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Agglutination tests Figure 35.12 titer = reciprocal of highest dilution positive for agglutination Figure 33.10a (top left): Tube agglutination test for determining antibody titer. The titer in this example is 160 since there is no agglutination in the next tube in the dilution series (1/320). The color in the dilution tubes indicates the presence of patient's serum. Figure 33.10b: A microtiter plate illustrating hemagglutination. The antibody is placed in the wells (1-10). Positive controls (row 11) and negative controls (row 12) are included. Red blood cells are added to each well. If sufficient antibody is present to agglutinate the cells, they sink as a mat to the bottom of the well. If insufficient antibody is present, they form a pellet at the bottom. Figure 35.12 Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Complement Fixation binding of complement to an antigen-antibody complex basis of diagnostic tests that determine if antibodies to an antigen are present in patient’s serum Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Figure 33. 11a: Test serum is added to one test tube Figure 33.11a: Test serum is added to one test tube. A fixed amount of antigen is then added to both tubes. If antibody is present in the test serum, immune complexes form. Figure 33.11b (middle): When complement is added, if immune complexes are present, they fix complement and consume it. Figure 33.11c (right): Indicator cells and a small amount of antierythrocyte antibody are added to the two tubes. If there is complement present, the indicator cells will lyse (a negative test); if the complement has been consumed (second step of test – middle drawing), no lysis will occur (a positive test). Figure 35.13 Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Enzyme-Linked Immunosorbent Assay ELISA can be used to detect antigens in a sample can be used to detect antibodies in a sample

Copyright © The McGraw-Hill Companies Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. Figure 35.14

Immunoblotting (Western Blot) procedure proteins separated by electrophoresis proteins transferred to nitrocellulose sheets protein bands visualized with enzyme-tagged antibodies sample uses distinguish microbes diagnostic tests determine prognosis for infectious disease Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Immunoblots of standard strains of Clostridium dificile Immunoblots of standard strains of Clostridium dificile. Arrows indicate strain-specific bands of the various proteins in different lanes (A-E). Molecular weights are indicated on the left. Figure 35.15 Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Immunoprecipitation used to detect soluble antigens binding of bivalent or multivalent antibodies to antigen forms lattice that precipitates lattice formation occurs only when there is an optimal ratio of antigen to antibody Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Figure 35.16 (left): graph showing that a precipitation curve is based on the ratio of antigen to antibody. The zone of equivalence represents that optimal ratio for precipitation. Figure 33.17b (right): a precipitation ring test. Antibodies and antigens diffuse toward each other in a test tube. A precipitation ring is formed at the zone of equivalence. Figure 35.16 Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Immunodiffusion precipitation reaction that occurs in agar gel medium two commonly used techniques single radial immunodiffusion (RID) assay double diffusion agar assay (Ouchterlony technique) Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

RID Figure 35.17 (a) used to quantify antigen Three standard solutions of different antigen concentrations and an unknown are placed on agar. After equilibration the ring diameters are measured. Usually the square of the diameter of the standard rings is plotted on the x-axis and the antigen concentration on the y-axis. From this standard curve, the concentration of an unknown can be determined. Figure 35.17 (a) Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Double diffusion agar assay precipitated immune complexes used to determine identity of antigen Figure 35.17 (b) Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Immunoelectrophoresis antigens first separated by electrophoresis according to charge antigens visualized by precipitation reaction has greater resolution than immunodiffusion assays Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Figure 33.15a: Antigens are separated in an agar gel by an electrical charge. Figure 33.15b: Antibody (antiserum) is then placed in a trough cut parallel to the direction of the antigen migration. Figure 33.15c: The antigens and antibodies diffuse through the agar and form precipitin arcs. Figure 33.15d: After staining, better visualization is possible. Figure 35.18 Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Flow Cytometry flow cytometry flow cytometer detects organisms in clinical samples detection based on cytometric parameters or by use of fluorochromes fluorochromes often bound to antibodies or oligonucleotides flow cytometer forces suspension of cells through laser beam and measures amount of light scattering of fluorescence can detect heterogeneous microbial populations with different responses to antimicrobial treatments Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Radioimmunoassay (RIA) purified antigen labeled with radioisotope competes with unlabeled standard for antibody binding amount of radioactivity associated with antibody is measured Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Susceptibility Testing testing for susceptibility to antimicrobial agents can be used to identify microbe particularly useful for determining proper therapy determined by: dilution susceptibility tests disk-diffusion tests (Kirby-Bauer method) E test Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.

Computers in Clinical Microbiology improve efficiency and increase speed and clarity with which results can be reported to physicians major uses test ordering result entry analysis of results report preparation also useful for lab management Copyright © The McGraw-Hill Companies. Permission required for reproduction or display.