ACP601: Plant Mycology and Bacteriology Plant Bacteriology

Transcription

1 ACP601: Plant Mycology and Bacteriology Plant Bacteriology James W. Muthomi Department of Plant Science and Crop Protection University of Nairobi

2 Bacterial Diseases of Plant in East Africa Bacterial blight of cotton

3 Bacterial Diseases of Plant in East Africa Bacterial blight of cassava (Xanthomonas axonopodis pv. manihotis)

4 Bacterial Diseases of Plant in East Africa Bacterial soft rot on pepper and carrot (Erwinia carotovora subsp. carotovora)

5 Bacterial Diseases of Plant in East Africa Angular leaf spot on cucumber (Pseudomonas lacrymans)

6 Bacterial Diseases of Plant in East Africa Banana Xanthomonas wilt (Xanthomonas campestris pv musacearum)

7 Bacterial Diseases of Plant in East Africa Halo blight of bean (Pseudomonas savastanoi pv phaseolicola)

8 Bacterial Diseases of Plant in East Africa Common bacterial blight of bean (Xanthomonas axonopodis pv phaseoli)

9 Bacterial Diseases of Plant in East Africa Black rot of cabbage: Bacterial wilt of potato: Bacterial wilt of potato and tomato (Ralstonia solanacearum)

10 Bacterial Diseases of Plant in East Africa Black rot of cabbage: (Xanthomonas campestris pv campestris)

11 Bacterial Diseases of Plant in East Africa Bacterial canker of tomato (Clavibacter michiganense subsp. michiganense)

12 Bacterial Diseases of Plant in East Africa Bacterial spot on tomato and pepper (Xanthomonas campestris pv. vesicatoria)

13 Bacterial Diseases of Plant in East Africa Crown gall of roses (Agrobacterium tumefasciens)

14 Bacterial Diseases of Plant in East Africa Citrus canker

15 Bacterial Diseases of Plant in East Africa Citrus greening disease (Candidatus liberobacter)

16 Morphology of Bacteria Morphology of Bacteria Cellular structure s Arrangement of cells Types flagellation Differences between Gram positive and Gram negative bacteria Examples of plant pathogenic bacteria

17 Morphology of Bacteria

18 Plant Pathogenic Bacteria Gram positive genera Clavibacter Curtobacterium Rathayibacter Streptomyces Gram negative genera Acidovorax Agrobacterium Burkholderia Enterobacter Erwinia Pantoea Pseudomonas Ralstonia Xanthomonas

19 Growth and Reproduction in Bacteria Bacterial growth curve and calculation of generation time Measurement of bacterial growth o Use of haemocytometer o Plate counting techniques (use of serial dilution) o Measurement of turbidity (spectrophotometer)

20 Isolation of Bacteria

21 Epidemiology of Bacterial diseases Sources of bacterial disease inoculum o Primary sources seed, planting materials, water, soil, crop debris, volunteer crops, weeds, insect vectors o Secondary sources human activities, rain splash Dissemination of bacterial diseases Entry into host plants o Natural openings stomata, lenticels, nectaries, hydathodes o Wounds Survival

22 Mechanisms of infection in Bacterial diseases Extracellular polysaccharides Pectic enzymes Toxins Plant growth regulators Others o Necrosis o Ammonia accumulation o Ice-nucleating proteins

23 Types of diseases caused by phytopathogenic bacteria Bacterial spots and blights Bacterial soft rots Bacterial vascular wilts Bacterial cankers Bacterial galls Bacterial scabs

24 Diagnosis of bacterial diseases of plants Preliminary diagnosis o Symptoms o Signs of bacteria streaming, ooze Isolation and cultural characteristics Gram staining Flagellation and mortility Physiological and biochemical tests o Oxidative/ fermentative tests o Levan production o Utilization of nitrogen compounds o Catalyse test o Starch hydrolysis o Salt tolerance

25 Diagnosis of Bacterial Diseases of Plants Water soaking

26 Diagnosis of Bacterial Diseases of Plants Bacterial ooze.umn.edu/distribution/horticulture/dg1169.html; ages/105/pests vision.org/default/ct/79/pests y/set/bacterial+wilt+protocol bacterial "ooze" or exudate seen coming out of water soaked lesions (see arrows). The "ooze" forms in the readily seen droplets. These droplets are a sign of the pathogen, being composed mostly of bacterial cells

27 Diagnosis of Bacterial Diseases of Plants Bacterial streaming test diagnosis of bacterial wilt To properly diagnose bacterial wilt, a horizontal cut was made in the lower stem and the cut stem immersed partway in water. Within a few minutes, copious amounts of bacterial exudate emerged from the cut end, forming the white streamers you see in the water. This only occurs with bacterial wilt and not with any other type of pathogen or abiotic cause

28 Diagnosis of Bacterial Diseases of Plants

29 Diagnosis of Bacterial Diseases of Plants Isolation of bacteria

30 Diagnosis of Bacterial Diseases of Plants Cultural characteristics Colony morphology

31 Diagnosis of Bacterial Diseases of Plants Colony morphology

32 Diagnosis of Bacterial Diseases of Plants Gram stain test KOH solubility test for Gram stain

33 Diagnosis of Bacterial Diseases of Plants Cell walls of Gram positive and Gram negative bacteria

34 Diagnosis of Bacterial Diseases of Plants Biochemical Tests Biochemical tests are used to identify bacteria to genus level i. Oxidation Test ii. Gas Production iii. Starch hydrolysis iv. Salt tolerance v. Catalyse test vi. Nitrate reduction test vii. Urease Activity viii. Gelatin hydrolysis

35 Diagnosis of bacterial diseases of plants Biochemical tests Oxidation / fermentation Test o In presence of oxygen, glucose is oxidized; in absence of oxygen glucose is fermented. o Oxidative (aerobic) bacteria metabolize glucose only in presence of oxygen to produce acid, resulting in ph change o Fermentative (anaerobic) bacteria metabolize glucose both in presence or absence of oxygen.

36 Diagnosis of bacterial diseases of plants Biochemical tests Gas Production obacteria metabolize carbohydrates to produce either acid alone, or acid together with gas. ogas production is tested by growing bacteria in a liquid medium to which a carbohydrate (glucose, sucrose, fructose, xylose, arabinose, mannital, sorbitol) has been added. A small tube is inverted into the medium. oif the bacterium utilizes the carbohydrate & produces a gas, the gas will collect in the small inverted tube

37 Diagnosis of bacterial diseases of plants Biochemical tests Tetrazolium salts tolerance test o certain bacteria are sensitive to tetrazolium salts. o To carry out the test, bacteria is spot inoculated onto a medium containing 0.02% and 0.1% triphenyl tetrazolium chloride (TTC) and incubated. o Tolerance to tetrazolium salt will be indicated by presence of growth. Salt tolerance test o bacteria is inoculated into nutrient broth containing required sodium chloride concentration. o Bacterial growth, seen as turbidity, which signifies sodium chloride tolerance.

38 Diagnosis of bacterial diseases of plants Biochemical tests Gelatin hydrolysis o The bacterium is grown by making a single streak in the middle of a solid medium containing gelatin. o Gelatin hydrolysis may be detected by a clear halo around colonies after addition of 10% potassium dichromate in hydrochloric acid.

39 Diagnosis of bacterial diseases of plants Biochemical tests Nitrate reduction test o Some oxidative bacteria have the ability to grow in anaerobic conditions rich in nitrate ions. o Nitrate medium is stab inoculated with bacteria and incubated fro 3-5 days until good growth has been obtained. o An equal volume of Follet and Ratcliff s reagent is added and shaken well. o Development of an orange or orange brown colour is a positive reaction, indicating the presence of nitrite.

40 Diagnosis of bacterial diseases of plants Biochemical tests Starch hydrolysis o A single streak of test bacterium is inoculated at the middle of starch-containing medium. o Incubate for 7 days, after which the plate is flooded with lugoli s iodine. o Starch hydrolysis is indicated by the presence of clear zone in the black-stained medium around or under the colonies. o Reddish zones indicate that starch has been partially hydrolyzed to dextrins, and is usually considered a negative reaction.

41 Diagnosis of bacterial diseases of plants Biochemical tests Utilization of urea o Bacterium is inoculated into ureacontaining medium and incubated for up to 7 days. o Urease activity is indicated by the development of a pink-red colouration. Urea test

42 Diagnosis of bacterial diseases of plants Biochemical tests Catalyse test omany oxidative (aerobic) bacteria produce enzyme catalyse that helps to protect them from toxic oxygen compounds ocatalyze regulates release of molecular oxygen from peroxides e.g. hydrogen peroxide. Test oa few drops of 3% hydrogen peroxide solution are placed onto a bacterial culture. oa positive result is indicated by the generation of oxygen bubbles (effervescence)

43 Diagnosis of Bacterial Diseases Serology ELISA Fatty acid analysis PCR-based analysis (molecular markers) Determination of pathogenicity o Inoculation of detached fruits o Stem inoculation for canker and gall inducing pathogens o Leaf inoculation for leaf spot pathogens o Cotyledon inoculation o Root inoculation o Inoculation of storage organs for soft rot bacteria

44 Diagnosis of bacterial diseases of plants Fatty acid analysis o Bacterial cell membranes contain many different fatty acids o Types and relative proportions of these are unique to a particular species. o Fatty acids are extracted & analyzed by gas chromatography and the fatty acid profiles are used to identify the bacteria genus, species, and, in some cases, to strain level

45 Diagnosis of Bacterial Diseases of Plants Gas Chromatogram fatty acid profiles

46 Diagnosis of bacterial diseases of plants DNA based analysis o Methods are based on gene sequences in the DNA of the bacterial chromosome. o Gene sequence is unique to a particular species or strain. o The steps in DNA analysis include: i. Bacterial DNA is first extracted from the cell ii. The extracted DNA is amplified by polymerase chain reaction (PCR), iii. The amplified DNA fragments are separated by electrophoresis on agarose gel to produce finger prints (DNA profiles). iv. Based on the banding pattern produced on the DNA profiles, the bacteria are identified by comparison to profiles of known DNA

47 Diagnosis of bacterial diseases of plants PCR analysis Nucleic acid-based analysis PCR analysis of bacteria

48 Diagnosis of bacterial diseases of plants Serology (ELISA) o A specific antibody is conjugated to polystyrene wells and a suspension of the test bacterium is placed in the well and allowed to react with the antibody. o A positive reaction is indicated by colour change, which can be detected by eye or measured by spectrophotometer (ELISA reader). Antigen-antibody reaction ELISA plate

49 Diagnosis of Bacterial Diseases of Plants Host inoculation tests Pathogenicity test Isolated bacteria Inoculate on germinated bean cotyledons Water soaking sympom

50 Management of bacterial diseases Sanitation Adjusting fertilization and watering Crop rotation Use of resistant varieties Soil sterilization use of clean seed and planting materials Use of chemicals Use of biological control agents