A Literature Review on

Seed Health Testing for Bacterial

lnfection




Compiled by:
Babu Ram Panthi
BScAg
IAAS, Rampur Campus
Rampur, Chitwan, Nepal
creation_babu@yahoo.com




Table Of Contents
S.No. Contents Page No.
1. Introduction 1
2. Objectives 9
3. Materials and Method 10
4. Literature review 10
5. Summary and Conclusion 12
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6. References 13
1. INTRODUCTION
Seed health
Seed heath refers mainly to the presence or absence of disease causing organism e.g. Fungi,
bacteria, viruses, insects pest and even the physiological condition, for trace element deficiency.
Seed health is one of the most important component of seed quality. Disease or pest infested seed
play a major role in the spread of diseases causing pathogens and pest from one place to another.
Fungi, bacteria, viruses, nematodes, eggs and /or larva of insect and even deficiency of micro
elements may cause diseases which may be transmitted to successive crops, affecting its yield
potentiality. Health seed should, therefore, be free from such diseases, insect pest, other
pathogenic organisms and deficiency symptoms. Te importance of detection and control of seed
borne diseases have been practiced for many years. Burning of wheat seed for controlling smut
or tossing of sorghum and sugarcane seeds on straw flame were practiced by farmers in the 18
th

century. Such indigenous method were gradually replaced by scientific methods and
technologies for the use of fungicides and pesticides as prophylactic treatment were developed to
prevent the occurrence of seed borne diseases. Production of healthy seeds, which dont carry
disease causing factors, help to maintain seed health. Seed health testing information is important
for the following reasons:
a) Seed borne microbes may give rise to disease development in the field and reduce
commercial value of the crop.
b) Imported seed lots may introduce new diseases and insect pests.
c) It may cause poor germination or field stand there by decimating yield.

Seed Borne Diseases
The term Seed Borne merely indicates that the pathogen and seed are associated in one
of the following ways.
Accompanying:- The pathogen may independently accompany the seed, without being attached
to it, for e.g. Ergot sclerotia smut balls etc.
External:- The pathogen may be carried passively on the surface of the seeds, such as, fungus
spores, nematode larvae or a virus.
Internal: - It is carried embedded in plant parts essential for the production of the seedlings, for
e.g. loose smut of wheat wherein the pathogen is imbedded in the embryo.
Seed Health Testing: Seed health testing is the science of determining the presence or absence
of disease causing agents, such as fungi, bacteria and viruses and animal pests such as eelworms
and insects in the seed samples. The extent of presence of disease infected seeds or the
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infestation by insect pests determines the seed heath status of a seed sample and by inference
seed lots.
Diagnostic Strategy
It is based for most diseases upon symptoms, isolation and practical identification schemes
using relatively few but important key tests. When testing for seed borne bacterial pathogen from
seeds, the following steps are usually involved:
1) Extraction of bacteria from seeds.
2) Isolation and purification.
3) Identification of the bacterial isolates.
Extraction of bacteria from seeds
Bacteria can be extracted from seeds by following methods:
1. Direct plating of seeds on various media followed by incubation at optimum conditions,
especially temperatures ranging between 25 and 30C, and darkness.
2. Soaking either whole seeds or ground-up seeds in sterile water or saline water for
different durations followed by plating the water containing bacterial cells onto different
media including semi selective or selective ones.
3. growing seedlings from seeds on various substrates and then isolating bacteria from plant
showing symptoms followed by the streaking onto a medium, preferably general ones
like Nutrient agar (NA), Growth factor (GF), KB etc
Isolation and purification of bacteria
Bacteria thus extracted are isolated on media of special composition and purified in order
to be able to identify them. The procedure is described below.
1. Surface sterilize infected plants by dipping into 95% ethanol before treating with NaOCl
(0.5-1%) solutions for 2-5 minutes and rinse 2-3 times with sterile water. Leaf materials
or roots can also be washed in running tap water for 1-2 hours.
2. Cut small area of the affected tissues on a clean glass slide and mount in a drop of water.
Put a cover slip and examine under compound microscope for streaming of bacterial
cells. Only if bacterial ooze is observed proceed with further steps of isolation.
3. Remove the cover slip and put the infected tissue in a few more drops of water, tease
apart he tissue and leave it for 10-15 minutes to obtain more bacterial cells in the water.
4. Using a bacteriological loop streak the water containing bacteria a number of times on a
suitable medium in a cross wise manner. Try to get single colony of bacterium.
5. Incubate the streaked plates in an inverted position at 25-30C.
6. Start observing the plates after 1-2 days for pseudomonas and up to 4 days for
xanthomonads. The colonies of pseudomonads on nutrient agar are white, or whitish
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grey. The colonies of xanthomonads on almost all the media are usually of creamy to
yellow color.
7. Purify the suspected single colonies by transferring them to a suitable medium. Flame the
loop in between streaking in order to obtain single colonies.

Identification
After obtaining pure cultures of the bacterium identification can start. Usually a
combination of the below described methods are used. These include: serological methods,
morphological and biochemical characters, pathogenicity tests, including the tobacco
hypersensitive test, electron microscopy, and others.

Major criteria used in identification
Morphological Characters
These include cell shape, size, capsule, endospores, intercellular inclusions, flagella and
colony characters such as color, margin size , elevation, consistency and others. Many diagnostic
and identification tests are based upon the nutritional and physiological properties of bacteria,
such as gram reaction, presence of cytochrome-c, pectate lyase activity, etc. in any case one
looks for overall similarities of the characters between isolates and the pattern described in text
books such as, Bergeys Manual of Determinative Bacteriology. Divergences by individual
isolates in the individual characters are usually disregarded. Identification based in such tests can
be time consuming, laborious and unsuitable for use at diagnostic laboratories as in routine seed
health testing for bacteria.
Serological tests
These tests have become an important tool in the diagnosis of many pathogens. Tests
like, slide agglutination (SA), Ouchterlony double diffusion (ODD), immunoflorescence (IF) and
enzyme linked immunosorbent assay (ELISA) are commonly applied in the identification work.

Inoculation Tests
Pathogenicity. A positive host test is often the most determinative character and usually
required to confirm diagnosis. Negative reactions or atypical symptoms should be taken with
caution.
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Hypersensitive reaction. Plant pathogenic bacteria are able to elicit a response in a non
host plant when injected in high numbers. The tst is usually carried out in tobacco leaves and
have proven of being valuable for most pseudomonads but not for all xanthomonads tested
(Lelliot and Stead, 1987)
Basic tests
Biochemical tests
Gram stain (Hucker modification).this reaction test is essential for differentiating
bacteria into two broad groups: gram positive and gram negative bacteria. The procedures are as
follows:
y Make a fairly turbid suspension of the testing bacterial growth from a solid medium in
sterile water. Include a positive and a negative control.
y Smear a loopful of the suspension on a slide.
y Air dry and passing the slide rapidly two or three times through a Bunsen flame.
y Flood the slide with crystal violet solution for 1 minute.
y Wash in gentle stream of tap water until no ore stain can be removed from the smear.
y Flood with Lugols iodine solution for 1 minute.
y Wash as in 5 and blot dry.
y Decolorize by washing in a gentle stream of ethanol(95%) for no more than 30 seconds to
remove stain that will easily wash away and blot dry.
y Counterstain by flooding with safari for 20 seconds.
y Wash with tap water, blot dry and examine by adding a drop of immersion oil and
observe under the compound microscope oil immension objective.
y Gram-negative bacteria stain pink red; gram-positive bacteria stain blue-violet.

Gram staining generally gives satisfactory result from the plant pathogen although older
cultures of Coryneform bacteria may stain gram-negative or variable. An alternative is the non-
staining Gram-reaction test, better known as the KOH solubility test which is as follows:
1. On a glass slide mix a loopful of bacteria from a well grown colony in a drop of 3% KOH
aqueous solution. Mixing should not exceed 10 seconds. A toothpick can be used instead
of loop.
2. Raise the loop or the toothpick a few centimeters from the glass slide. If strands of viscid
material are seen, the bacterium in question is gram-negative. Gram-positive bacteria do
not produce such strands even on repeated strokes.
Pigmentation. Plant pathogenic bacteria produce a variety of pigments, some of which
are made manifest themselves only on special media. Pseudomonas species produce several
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kinds of pigments which are of taxonomic and diagnostic value. One group of diffusible
pigments consists of the fluorescence types which are produced by so-called fluorescent
pseodomonads. The green diffusible fluorescent pigments are best produced in media of low iron
content. The most widely used medium for this purpose is Kings medium B. the fluorescence
can readily be seen under ultraviolet light.
Yellow carotenoid pigments are produced by Xanthomonas Spp., Erwinia herbicola is
also known to produce similar pigmentation. When the suspected colonies are purified and used
further in pathogenicity tests only the pathogenic xanthomonads will produce symptoms in
inoculated plants.
Arginine dihydrolase (Lelliott and Stead, 1987). The arginine dihydrolase complex
system permits certain pseudomonads to grow under anaerobic conditions. Ammonia is evolved
which brings about the change in pH, indicating a positive reaction. The procedures are as
follows:
1. Inoculate by stabbing with a fresh culture tube containing 3 ml of Thrnleys medium.
2. Cover with sterile mineral oil or molten Vaseline.
3. Incubate for 3 days at 27C.
4. A change of color to red is a positive reaction.
Levan (poly-fructose) formation. Pseudomonas syringae produces white, domed,
shining, mucoid, levan type colonies on a5% sucrose nutrient agar. isolated colonies are usually
3-5mm after 3 days at 27C.
Levan, formed by levan sucrose, is produced by most pseudomonads which utilize
sucrose. The reaction is useful in the identification of pseudomonads.
Inoculate plates in order to obtain separate colonies when testing pure cultures. Serial
dilutions from infected samples or seed extracts are similarly used for the inoculation of the
plates.
Kovacs oxidase test (Kovacs, 1956; Hildebrand and Schroth, 1972). The test is of
particular differential value within the genus Pseudomonas. It can be useful to distinguish the
complex of species related to P. syringae, which are oxidase negative from other oxidase positive
pseudomonads.
1. Place a Whatman filter paper No. 1 in a petri dish and add 3-4 drops of freshly prepared
1% aqueous solution of tetramethylparaphenylene-diamine dihydrochloride on the centre
of the paper.
2. With the help of a platinum loop, take a loopful of bacteria, grown on Kings medium B
and streak onto the moist filter paper.
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3. Change of the regent to a purple color within 10 seconds of application of the culture
should be regarded as a positive test result.
Note: Since Tetramethyl-paraphenylene-diamine dihydrochloride is a rather expensive reagent,
prepare the solutions only in the required amount to be used. The reagent can also be made
weekly or fortnightly and stored in a stoppered dark glass bottle at 4C. Care should be taken to
avoid contact of the powder or solution with the skin.
Lipase activity (Lelliott and Stead, 1987). The procedure is as follows:
1. Inoculate Tween 80 agar plates and try to obtain single colonies.
2. Incubate for up to 7 days.
3. Development of a milky-white precipitate around the colonies demonstrate the ability to
hydrolyse the lipid Tween 80.
Starch hydrolysis (Lelliott and Stead, 1987). The procedure is as follows:
1. Inoculate starch agar plates by streaking and incubate for at least 4 days.
2. Flood with Lugols iodine.
Results: negative the medium will turn blue if starch is not hydrolysed. Positive Yellowish,
clear zones appear around or under the bacterial growth. Reddish colored zones indicate that
starch is partly hydrolyzed to dextrin and is usually considered a negative reaction.
Nitrate Reduction test (Lelliott and Stead, 1987). Some oxidative bacteria, including
many pseudomonads, have the ability to grow under anaerobic conditions rich in nitrate ions.
Energy is derived by reducing nitrate to nitrite. Since nitrate is toxic some bacteria further nitrite
to nitrous oxide and/or gaseous nitrogen by the nitrite reeducates system.
Nitrate reeducates nitrite reeducates
NO
3
----------------------- NO
2
-------------------- NH
3

1. Stab inoculate the nitrate medium in duplicate for 3-5 days. Include an uninoculated test
tube.
2. Add equal volume of Ratcliffs reagent to one inoculated and uninoculated control at 3
and 5 days.
Shake well.
Development of orage to orange brown colour means positive (nitrite present)
No color development within 10 minutes add a pinch of cadmium powder.
No color development after 10 minutes: Positive (nitrate reduction beyond nitrite)
The uninoculated control should develop the color in the presence of reagent and the
metal.

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Utilization of carbon compounds (Lelliott and Stead, 1987). This is the test usually
referred to the ability to test organism to utilize a compound as the source of carbon and of
chemical energy. Utilization is either demonstrated by the presence of growth or by a biological
change brought about in the medium (acid alkali shift demonstrated by the use of the suitable
indicator). The test can also be used to determine whether a bacterium has a respiratory or
fermentive metabolism (creating anaerobic conditions: covering with sterile mineral oil). All
known plant pathogenic bacteria except Clostridium spp are facultative anaerobes and have a
fermentative metabolism.

Carbohydrate and organic acid utilization base. A synthetic base containing no
organic nutrients may be used for the tests of the non fluorescent and fluorescent pseudomonads.
1. Prepare carbohydrate solutions filter sterilized (neutralized if necessary).
2. Add to the sterile base (molten, if necessary at 45C) to obtain the following
concentrations:
a. Non-fluorescent pseudomonads-liquid base; sugars at 0.2%, other carbohydrate at 0.1%.
b. Fluorescent pseudomonads-agar base; sugars at 0.5%, other carbohydrates at 0.1%.
3. Inoculate the medium and incubate.
4. Record presence or absence of growth.

Pathogenicity tests
The aim of a host test is to determine whether or not a suspected pathogen can produce
disease symptoms in the host from which it was isolated. For many plant diseases this test is the
only method for confirming or negating an initial presumptive diagnosis. The following criteria
are usually followed.
1. The pathogen is introduced into or on to the plant in a manner that mimics the natural
infection as closely as possible.
2. The cell concentrations used should be similar to those concentrations that cause natural
infections or at least do not exceed natural inoculums levels.
3. Cultivate the pathogen under conditions which do not seriously affect virulence.
4. Use plants which are naturally susceptible to infection and in conditions in which
infection can occur naturally.
5. Incubate the inoculated plant under natural growing conditions.
6. Determine what can be considered as typical symptoms.
Preparation of the bacterial suspension. (Kiraly et al., 1970). A 24-48 hours slant agar
culture is used. The agar culture is washed with sterile water or loopful of bacterial growth are
resuspended in nutrient broth, sterile water or saline, and the suspended is adjusted to a
determined bacterial cell number.
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Inoculation. The inoculation methods are as follows:
Spraying plants. This involved misting inoculums at comparatively low concentrations
on to plants. A few leaves and/or stems of which can be lightly wounded with pricking needles.
Plants are incubated at high humidity.
1. Young plants are incubated in polythene bags with wires arranged to avoid the bags
touching the plant, for 24 hours before inoculation.
2. Inoculums is prepared in concentration of 10
5
- 10
6
cfu/ml.
3. Prick one or two labeled leaves on each plant and the upper part of the young stem in
about five pieces with a sterile needle.
4. Spray inoculums as a fine mist with a low pressure sprayer until the plant surface is wet,
ensuring that upper and lower leaf surfaces are wetted. If a high pressure atomizer is used
no more than 10
6
cfu/ml should be used. Plants can be inoculated while still inside the
polythene bags, reducing the risk of infecting other plants.
5. Close the polythene bag with inoculated plants incubate for 48 hours. Include a positive
control by inoculating plant with a known culture and negative control by spraying just
with sterile saline or water.
6. After incubation transfer the plants in a glasshouse or growth chamber at 25-30C and
high relative humidity with 12-16 hours light.
7. Observe periodically for symptoms; water soaked lesions developing from three days
onwards. These could become necrotic, usually brown black and with water soaked
margins along the veins. On stem lesions are often elongated. If stomata infection do not
occur wound infections usually succeed in producing symptoms, but caution is then
needed to interpret the results.

Hyper sensitive test ( Lelliott and Stead, 1987)
Most bacterial plant pathogens can induce a hypersensitive response when injected into
the tissues of a non- host plant. Non pathogenic bacteria and some plant pathogens, particularly
those that are opportunistic or gall forming, do not inside this hypersensitive response. The
reaction can provide an extremely useful presumptive diagnostic technique.
A variety of plant may be used, but for many bacteria tobacco is the preferred plant, since
it is easy to cultivate and maintain. Its large cavity beside leaf vein makes it relatively easy to
infiltrate the inoculums and its reaction to many pathogens is well known. But for xanthomonads
tomato is preferred.
Serological test
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Serological test has become an important tool in the diagnosis in plant pathogenic
bacteria. There are different serological tools including enzyme linked immunosorbent assay
(ELISA) practiced in seed health testing for bacteria. Here, agglutination test, the commonly
practiced in seed borne bacterial pathogens described.
With this test a positive reaction is given by agglutination of bacterial cells due to
multiple bridging between specific antibodies and antigens of the cell wall and flagella.
Agglutination can be seen by naked eye but weak agglutination is best seen under a low
magnification stereo-microscope.
Before agglutination test are made the optimal dilution point of the antiserum where
definite agglutination occurs most be established. This is done by preparing serial dilution of the
antiserum in 0.85 % Nacl and testing these dilutions in test tubes by adding an equal volume of
the bacterial suspension prepared in 0.85% Nail or 0.01 M buffer solution. The test tube should
be shaken a couple of times gently in half clockwise and half anticlockwise and later incubated at
37
0
c for 2 hrs. Record carefully the last dilution where agglutination is observed. The working
dilution of the antiserum is two steps lower than the titre of the antiserum. The working dilution
of the given antiserum is the one which should be used in the routine seed health test. When the
test is performed in test tubes, it is called Tube agglutination test and Slide agglutination when
the test is performed on glass slides.

2. OBJECTIVES
1) To determine their quality that is their suitability for planting.
2) To identify seed quality problems and their probable cause.
3) To determine the need for drying and processing and specific procedures that should be
used.
4) To determine if seed meets established quality standards or labeling specifications.
5) To establish quality and provide a basis for price and consumer discrimination among
lots in the market.
6) To determine the health status of the seed lot which in turn establishes the sanitary
conditions of the seed in commerce.
7) To obtain objective proof of whether the seed lot meets the requisite certification
standard or not.
8) To obtain objective proof of whether the lot meets the requisite quarantine requirement.
9) Occasionally, health test may also be made for tracing the cause of low germinating
capacity or poor field emergence due to diseases or insect pests.



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3. MATERIALS AND METHOD:
Materials:
Books of national and international writers
Journals published by national and international organizations
Thesis papers of M.Sc. Ag and Ph.D. in Plant pathology and plant protection
Internet
Class note of P.P. Pandey and Bhim Chaulagain.
Place of search:
IAAS Central library
Internet
Lecture of plant pathology
For the completion of my assignment I search through out the library for the related books. Plant
pathology books and related references were helpful for the completion of my assignment. As my
topic is for seed health testing for bacterial infection, I give emphasis for the books of Seed Health
in library. Besides library work I search my topic through the net and acquired my related topics
there. Lecturer notes provided by class teacher was very much helpful.


4. LITERATURE REVIEW
Methods of detecting seed borne bacterial plant pathogens ( Rattan Lal Agrawal,1999)
This is relatively difficult area of seed borne diseases. The following approach may be adopted:
1) Examination of external characteristics visible on the seed coat of infected seeds.
2) Examination of disease symptoms on growing plants.
3) Isolation of the bacterial pathogen and its identification using biological or serological
tests.
4) A combination of any of the above.

Over the years, various methods have been developed. Some of these methods are simple while
others are highly specialized methods namely, serological methods (double diffusion technique,
agglutination methods, immunoflourescence technique) and phage plaque methods.
Most of the plant pathogens bacteria are seed borne in nature. Plant pathogenic do not form
endospores with the exception of Agrobacterium , corynebacterium & Streptomyces , which are
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soil inhabitants. Most of the members of other genera arenot fit for the survival in soil. They largely
depend on their hosts for survival through critical seasons. Generally, they are soil invaders as well
as in vegetatively propagated hosts. The bacteria survive in infected tissues, in galls
(Agrobacterium tumefaciens ) in tubers of potatoes ( corynebacterium sependonicum ) in roots of
Lucern ( C. insidiosum) and in lesions in the bark of trees such as plum (xanthomonads monus
prunil) and plants on the other hand, the bacteria are more dependent on seed transmission and it is
of particularly importance in cultivated annuals such as phaseolus bean , soyabean, peas,
cucumber,cabbage, cotton , tomato and cereals.(Neerguard, 1979)
The techniques for the detection of plant pathogenic bacteria (P.K. Agrawal and M. Dadlani,1992)
The bacteria are often carried on the surface of the seed but those causing vascular or
systematic infections are frequently found in the seed coat or other tissues of the seed. For instance,
Pseudomonas phaseolicola and Xanthomonas phaseolia. In phaseolus bean found in the hilum
region of the seed into which they penetrate from the vascular systems through the funiculus.
Bacteria born on the surface of the seed may keep alive for a limited period of time only
perhaps one or two years, whereas the bacteria harbor within seed tissues may show a surprisingly
extended longevity, such as C. flaccumfaciens which can survive in phaseolus bean seeds for 24
years kept under lab conditions.
The techniques for the detection of plant pathogenic bacteria are comparatively less developed.
Routine seed health testing techniques for the detection of only two bacteria i.e. Xanthomonas
malvacearum in cotton and X. campestris in brassica seeds have been included in ISTA handbook
on seed health testing published in 1981 & 1982. The detection of rest of bacterial pathogen is
carried out in different ways in different lab in the absence of any standard technique approved by
ISTA.
( Alice m. Anderson and Charles m. Leach, 1961) Seed borne pathogen affects directly and
indirectly the quality of seeds in commerce of primary importance is the fact that the seed borne
plant pathogen introduces diseases such as smuts into newly sown crops thereby causing a reduction
in yield and its quality. The field germination of seed infected with seed borne pathogens may be
reduced because the pathogen attack and kill the seedlings.
Seed infected with bacterial pathogens generally are not distinguishable from healthy seeds.
To detect seeds infected with bacteria two main methods are available. The first is to grow plant in
the lab or greenhouse under optimum condition for the development of the pathogens until disease
symptoms are evident. A second method is rapid phage plaque count technique. It employs
bacteriophages capable of lysing or dissolving specific bacterial pathogens.


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5. SUMMARY AND CONCLUSION:
This assignment has encouraged us to search more and more about any single topic from various
sources available. Really, literature review section in this assignment taught me how to filter the
required fact from large collected materials. It also helped me to present in any subject matter
given.
Secondarily, by the overall study or project of this assignment writing on topic Seed health testing
for bacterial infection I became aware of its techniques used for the process of testing seed. It has
helped us to know about the health status of the seed lot which in turn establishes the sanitary
conditions of the seed in commerce.
Finally I become able to know the test methods involved in the seed testing for bacterial infection
and the control or preventive measures applied for the better seed health. This assignment has really
made be efficient in preparing the assignment.




















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REFERENCES

Mathur, S.B., P. Amatya K. Shrestha and H.K. Manandhar 1992. Plant Diseases Seed
Production and Seed Health Testing in Nepal, Danish Government Institute of Seed
Pathogoly for Developing Countries.

Sen, S. and N. Gosh, 1999. Seed Science and Technology, Kalyani Publishers.

Agrawal, R.L. 1999. Seed Technology, Oxford and IBH Publishing Co. PVT. LTD.- New
Delhi, Calcutta

Basra, A.S. 2002. Seed Quality- Basic Mechanism and Agricultural Implication, CBS
Publishers and Distributors.

Agrawal, P.K. and M. Dadlani. 1992. Techniques in Seed Science and Technology, South
Asian Publishers.

Clark, M.F. and Adams, A.N. 1977. Characteristics of the microplate method of enzyme-
linked immunosorbent assay for the detection of plant viruses. Journal of General
Virology
Hildebrand, D.C. and Scroth, M.N. 1972. Identification of the fluorescent pseudomonads. In
proceedings of the Third International Conference on Plant Pathogenic Bacteria,
Wageningen, Centre for Agricultural Publishing and Documentation.
Kiraly, Z.., Klement, z., Solymosy, F. and Voros, J. 1970. Methods in plant Virology,
Akademia Kiado, Budapest.
Kovacs, N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature,
London.
Lelliott, R.A. and Stead, D.E. 1987. Methods for the Diagnosis of bacterial Diseases of
Plants. Methods in Plant Pathology Vol. 2, ed. T.F. Preece. Blackwell Scientific
Publications.
Schaad, N.W. (ed.). 1988. Laboratory Guide foIdentification of Plant Pathogenic Bacteria,
second edition.APS press.