Reprinted from The Australian Journal of Science, Vol. 20, No. 3, October, 1957, p. 71.

Symposium on Bacterial and
Viral Genetics

 

A symposium on bacterial and viral genetics,
organized by the Department of Microbiology
of the Australian National University, was held
in the new Lecture Theatre of the John Curtin
School of Medical Research on Monday August
19 1957. It was supported in part by a grant
from the Australian Academy of Science, and
was attended by members of the Genetics
Society of Australia arid by microbiologists.
There were visitors from all States as well as
from England and U.S.A.

CATEGORY.
1. Heterokaryosis
2. Sexuality Fusion

3. Transduction
(a) DNA mediated
(b) Phage mediated
i. Generalized

ii Prophage-linked
lii. Prophage

Unir or EXcHANGE.
Intact nucleus

nucleus
genome or major part)
Genotypic fragment

Genetics of Vaccinia Virus: paper by Pro-

fessor Frank Fenner.
Exchange of Character Determinants between

Strains of Plant Viruses: paper by Dr. R. J.
Best.

Introduction
JosHuA LrDERBERG*

In genetic analysis, recombinational phe-
nomena play a central role. In order to explore
the genetic material, we must expose it to
experimentally defined reagents. Ultimately,
we shall learn more from chemical reagents,
but at present the most fruitful experiments in

EXAMPLES.
Streptomyces griseus.

(entire Escherichia coli, Pseudo-
monas aeruginosa, Strepto-

muyces coelicolor.
pneumococcus; Hemophilus.

Salmonella; £.
Pl.

£. coli (lambda-Gal).

coli: phage

(= lysogeny or lysogenic

conversion)

A summary of the papers delivered follows.
In the evening there was a general discussion
which was introduced by Dr. Joklik’s des-
cription of the application of autoradiography
to problems of DNA _ replication (‘astro-
biology’), and was concerned largely with
speculation on the chemical basis of heredity.

Introduction: paper by Professor Lederberg.

Genetic Recombination in Pseudomonas
aeruginosa: paper by Dr. Holloway.

in Esche-
Esther M.

Lysogenicity and Transduction
richia coli K12: paper by Dr.
Lederberg.

Genetics of Influenza Virus:
Macfarlane Burnet.

paper by Sir

genetics involve the reactions of genes with
one another, that is, recombination experiments,

Enough is now known of the genetic systems
of different bacteria to permit a_ tentative
classification in terms of the quality of the unit
subject to exchange:

More ean be said about each of these cate-
gories. In the #&. coli sexual system, for
example, Jacob has forwarded evidence that
the act of mating may be interrupted by

blending the bacterial suspension, so that
fertilization is incomplete. He and _ his
colleagues have reported the most. skilful

experiments which correlate the number of

* Department of Medicai Genetics, University of
Wisconsin. (Fulbright Visiting Professor of Bac-
teriology, University of Melbourne.)

 
72 THE AUSTRALIAN JOURNAL OF SCIENCE

OCTOBER

 

genes from the male parent which appear in a
sexual progeny, and the amount of “P-labelled
DNA which it contains. Other evidence suggests
that some matings, if left undisturbed, proceed
to virtually complete fertilization, and there is
still much to be learned in the quantitative
evaluation of the various steps: the extent of
initial transfer, the efficiency and uniformity of
pairing, and especially the mechanism of
‘incorporation’ or crossing-over.

Dr. Holloway will bring us up to date on his
studies of the Pseudomonas system, and Dr.
E. M. Lederberg will elaborate on the prophage-
linked transduction of Gal markers in £. coli.

The pioneering work on recombination in
animal and plant viruses is a distinctively
Australian contribution, and I am looking
forward to the papers of Sir MacFarlane

Burnet, Professor Fenner and Dr. Best for
my own information on these fascinating
developments,

Genetic Recombination in Pseudomonas
aeruginosa
B. W. Hottoway*

In view of the unusual nature of the genetic
processes that occur in bacteria it is desirable
that a range of bacteria be studied in order to
determine the generality of the mechanisms.
The techniques developed by Lederberg for
#. coli are applicable to certain other bacteria
and have proved successful with Pseudomonas

TABLE I

The Formation of Prototrophs in Crosses between Auxotrophs
in Four Strains of Pseudomonas aeruginosa

 

Strain I L 3 29

 

 

 

H+
I+
Leb

 

 

 

-+- = recombination.
+ = recombination at low frequency.
— = no recombination.

aeruginosa, a Gram-negative bacillus character-
ized by the formation of blue and green
pigments.

P. aeruginosa will grow on a medium con-
sisting of glucose and mineral salts. Auxo-
trophic mutants for a range of amino acids,
purines and pyrimidines were isolated using
manganous chloride as the mutagen. In certain
cases crosses between different auxotrophic
mutants resulted in the formation of recom-
binant prototrophs. The interfertility of the
four strains used is shown in Table I,

Different mutant combinations of any two
fertile parents gave different recombination

 

* Department of Bacteriology, University of Mel-
bourne.

frequencies of prototrophs (for example see
Table II).

Segregation of various non-selective markers
has also been shown and the frequent formation
of novel combinations of non-selective markers
provides conclusive evidence of some genetic
mechanism.

For example, in the cross I (T,IV,*S*Ct)—
a trytophane requiring, streptomycin sensitive,
chloramphenicol resistant mutant of strain I—
xL (IVrT,'S'C*)—an isoleucine plus valine
requiring, streptomycin resistant, chloram-
phenicol sensitive mutant of strain L—the
formation of recombinants which are geno-
typically T+ IV* S'C* can only be explained by
genetic reassortment of the marker genes.

TABLE IL

Recombination Frequencies of I (LIL)
with various Auxotrophs of Strain L

 

|

 

| Recombinants/10*

Cross total parental
cells
|
I(LIL-)xL(T,) .. 20

xL (Ad) .. 55
xL(Thr-).. 1
xL(Iv~) .. 200

 

I(LIL~)=auxotroph of strain I requiring
leucine.

T =tryptophane.

Ad =adenine

Thr = threonine,

IV =isoleucine and valine.

There is considerable evidence that the
mechanism of recombination is very similar to
that occurring in EH. coli. In both organisms
actual contact of the parental bacteria is
necessary for genetic recombination to take
place. There is a correlation between the F
fertility system in EZ. coli and the interfertility
of the various strains shown in Table 1. For
the combination I x L, with which much work
has been done, equation of strain I as the F-
and strain L as the F* gives a situation
identical with that of #. coli. In both organisms
there is an unequal contribution of the parents
to the recombinant, the F- parent contributing
more than the F*. Thus the recombinants tend
to resemble more the F- parent. Strain I and
strain L differ in their pigment forming ability,
colonial morphology and in their reactions to a
group of bacteriophages. 99 per cent. of
recombinants resemble the strain I parent in
these characteristics. Irradiation with ultra-
violet light of the F* strain of both organisms
increases recombination frequency, whereas no
such effect occurs with the F- strain. In EF. coli
the F factor is infectious being easily trans-
ferred from a F* to a F~ strain. Infectious
transfer of this nature has been shown for
Pseudomonas in preliminary experiments at
low frequency. The paucity of suitable markers
in P. aeruginosa has limited the linkage
1957

THE AUSTRALIAN JOURNAL OF SCIENCE 73

 

studies. It has been possible to divide the
available markers into two groups, those which
are inherited freely from the F* parent by the
F- parent and those which fail to be inherited
in this fashion. On the assumption of linearity
it has been possible to establish a provisional
chromosome map of the former group. Little
is known yet of the actual processes of conju-
gation and genetic transfer in Pseudomonas.

Current work with P. aeruginosa is aimed
at learning more of the actual mechanism of
genetic transfer and also the manner of the
inheritance of prophages from a multi-lysogenic
strain which has been derived from strain L.

Lysogenicity and Transduction in

Escherichia coli K-12
EsTHER M. LEpERBERG*

The relationship termed lysogenicity is
characteristic of bacteria which harbor viruses
or phages in a latent form. Such temperate
phages can undergo either a lytic cycle whereby
the host is destroyed immediately or can allow
the production of a viable bacterial clone which
liberates the phage sporadically over an
indefinite number of generations. This process
occurs stochastically in an untreated popu-
lation, and may be greatly accelerated by
mutagenic agents.

The starting point of genetic analysis of
lysogenicity is its transmission in the non-
virulent stage, prophage, during vegetative
reproduction as part of the hereditary consti-
tution of each cell. The accidental discovery of
lysogenicity in H. cdéli K-12 in which sexual
recombination had been demonstrated furnished
a unique opportunity to study its genetic basis.

The demonstration of lambda, the temperate
phage carried by strain K-12 became possible
when a rare mutation leading to loss of pro-
phage had occurred. This was associated with
loss of immunity and thus susceptibility to lysis
by lambda. The main phenotypes include Lp*
(lysogenic), Lps (sensitive), and Lpr
(immune). The latter carried a defective pro-
phage, conferring immunity or resistance to
lysis, but no phage active against Lps is
produced.

Intercrosses in every combination of these
types resulted in a strict segregation of the
ensuing progeny into just the parental classes,
suggesting the existence of an Lp locus for the
maintenance of lambda prophage at a specific
site on the chromosome. The chromosomal
hypothesis was strengthened by linkage tests
in crosses involving a number of genetic
markers: a close linkage of Lp to a cluster of
loci controlling galactose fermentation (Gal)
was found. The linkage was verified by the
linked segregation of Lp and Gal from hetero-
zygous diploids of several types. Segregation
patterns were also followed in homozygous
diploids: Gal-Lp*/Gal-Lp* which were first
infected with lambda and then allowed to

 

* Dniversity of Wisconsin.

revert to Gal*, The infected diploids were
lysogenic, showing that Lp* was dominant to
Lp’, but both were recovered as haploid segre-
gants. Thus the fixation of the prophage had
occurred on one of the two chromosomes. Gal*
reversions were then selected under the
appropriate conditions. The diploid then segre-
gated Gal‘/Gal- as well as Lp*/Lp*. The
reversion had occurred either on’ the Lp*—or
Lp*—marked chromosome (coupling phase or
cisdiploid vs repulsion phase or trans diploid).
A few crossover recombinants were also
identified.

On one hypothesis lysogenicity is determined
by the cytoplasm. It would be expected that
the diploid cell would provide the most
favorable opportunity for the intermingling
of the cytoplasm and would be detected by the
uniformity of the haploid segregants with
regard to their lysogenic character. However,
the segregation of Lp and Gal as an intact part
of the linkage group confirms the alternative
hypothesis of integration of lambda prophage
into a definite site on the chromosome.

Another link between phage and bacterial
genes is the process of genetic transduction
which is defined as the transfer of a fragment
of the genotype in contrast to sexual
recombination which involves an essentially
intact nucleus. The transduction mediated by
lambda differs from the generalized trans-
duction described for Salmonella in several
respects: (1) effective transducing lambda
arises only by induction of Lpt bacteria, not
by lytic growth on Lp’; (2) the transfer is
confined to the Gal loci linked to Lp; (3) the
transduction clone is unstable: a partial diploid
or heterogenetic stage continues to segregate
Gal+/Gal- but no other markers; (4) the
recipient may be lysogenic, immune, or
sensitive, providing that lambda is adsorbed;
(5) lysates produced from heterogenotes are
HFT, ie., every particle is potentially capable
of initiating a transductional event, in contrast
to LFT lysates (‘low frequency of trans-
duction’) from haploid donors.

The donor bacterium can be marked by a
genetically different Gal (a*b-) than the
recipient (a-b*). A Gal’ heterogenote (a-b*/ex
a‘b-) is formed and segregates in order of
decreasing frequency, recipient or endogenetic
type (abt), donor or exogenotic type (atb-)
and double Gal- crossover type (ab-). Re-
arrangement in a neterogenotic cell sometimes
results in homogenotes carrying the same Gal
allele on the exogenote and endogenote. Specific
Gal- HFT lysates may be produced from these
and exploited in homology or allelism tests.
Each Gal culture is transformed to Galt by the
HFT lambda from Galt or any nonallelic Gal-
donor. The results were fully consistent with
those from standard crossing experiments.

The ten distinctive loci controlling the
fermentation of galactose so far identified had
been isolated as unique mutational events.
Certain combinations of Gal- formed pheno-
typically mutant heterogenotes (a-b*/a‘b-).
74 THE AUSTRALIAN JOURNAL OF SCIENCE

OCTOBER

 

Rearranged heterogenotes of the constitution:
atbt/a-b- were phenotypically Galt, indicating a
cis-trans position effect. Combinations of a
mutant from one position-effect group or cistron
with one from the other are Gal‘; within the

cistron, the heterogenotes are Gal-. Two
position-effect groups have been identified
genetically and their enzymatic defect

determined: Gal 2, 8, 10 mutants are defective
in galactokinase; Gal 1, 4, 6, 7, 11, in UDP-
hexose-transferase. The latter carry the same
defect as galactosemia in man. The enzymology
of a third group, (Gal 8, 9), which give
position-effects with either of the other groups
is not yet known.

A close correlation of transduction and
lysogenization was observed. From Lp* re-
cipients either pure Lpt or Lp** heterogenotes
are found. The latter are nonlysogenic, immune
to lambda, but every segregant is Lp*. Several
hypotheses advanced to account for them are
under critical test. Transductions involving
the addition of prophages related to lambda as
other markers for the recipient show the
following significant features: (1)  hetero-
genotes segregaie lysogenic/sensitive for either
or both prophages along with two Gal loci;
(2) linkage of Gal type and lambda marker;
(3) continued segregation of one or both
prophages in segregants already pure for Gal.
Super-infection of a lysogenic with related
phages resulted in either unstable double
lysogenics or prophage substitution.

In the light of these findings lysogenization
may be regarded as a more restricted form of
transduction: the phage behaves as the vector
as well as the passenger in the transduction of
lysogenicity.

Genetics of Influenza Virus
F, M. Burnet*® .

For the past seven years extensive studies
on the genetic interaction of strains of influenza
virus have been carried out at the Hall Insti-
tute. As a preliminary to any work on
recombination it is necessary to establish the
conditions needed to isolate and maintain pure
clones of virus. The use of limiting infective
dilutions for this purpose was first worked out
in relation to the changes which take place
when an influenza virus is adapted to growth in
chick embryo (the O-D phenomenon). The
general principles involved in the appearance
of mutants and the selective survival of mutant
clones have been studied in a number of papers,
notably the investigations of Edney (1957) on
the progressive replacement of an original
strain by serum-resistant variants in the
presence of homologous immune serum.

For any type of genetic work it is necessary
to have clearly defined differences between the
forms used for hybridization and in general
it is helpful to have available a range of strains
showing many ‘marker’ characters. In viruses
jit is particularly valuable to have marker
difference which can be detected by simple

 

* Walter and Eliza Hall Institute, Melbourne.

in vitro tests. Many such characters can be
defined for influenza virus strains by using the
haemagglutinating activity of the virus as an
index. For all practical purposes the most
important character of a virus is its virulence
for its natural and laboratory hosts and another
series of marker differences will be based on
virulence for various host tissues or cells.

In our work the following markers have been
employed:
I. Based on haemagglutination
Aa Serological character
Bb Heat stability of haemagglutinin
Ce Inhibition of heated haemagglutinin
by ovomucin, ete.

Dd Inhibition of heated haemagglutinin
by sheep salivary mucin.

Il. Virulence characters
Ee Capacity to produce haemorrhagic
lesions in chick embryo.
Ff Extent of lung lesions after intra-
nasal inoculation of mice.
Gg Pathogenicity on intracerebral inocu-
lation in mice.

III. Morphology

Hh Proportion of filaments in standard
infective fluid.

When conditions are arranged so that sus-
ceptible cells can be simultaneously infected
with two different influenza virus strains, the
following types of genetic interaction may be
recognized: .

1. Transfer of neurotropism from NWS to
other serological types of influenza A
(Burnet and Lind, 1949).

2. Reciprocal recombination within the

MEL/WSE system with definition of 2

linkage groups’.

Redistribution of virulence in

systems, not in others.

4. Phenomenon of double neutralization —
phenotypic mixture.

5. ‘Resurrection’ of virus killed by heat or
UV by appropriate recombination.

6. Phenomenon of heterozygosis.

Recent work has been concerned largely with
a phenomenon which seems to involve the
action of a suppressor gene. A _ strain of
influenza virus, WSE, highly virulent for mice,
gave rise to a mutant completely lacking in
virulence. This avirulent strain, aWS—, was
genetically stable and could be passed without
change in any of its properties indefinitely at
limit dilution. If, however, passage from one
embryo to another is made by inoculation of
the allantoic cavity with relatively concentrated
infective fluid, mouse virulent mutants
appeared after a few generations. Analysis of
the process indicates that the reversion to
virulence takes place by a series of steps. It
is possible to obtain revertants to full mouse
virulence without the necessity of passage

some

oo
1957

THE AUSTRALIAN JOURNAL OF SCIENCE / 1

 

through mice. It seems evident therefore, that
there has been no true genetic loss of virulence
in the change from WS-— to aWS— and some
form of masking or suppressor effect must be
postulated.

The problem of stabilizing virulence or lack
of virulence in virus strains is often a matter
of great practical importance. The existence
of strains of the aWS— type showing masked
virulence indicates one of the major difficulties
in being sure that any attenuated variant has
been ‘fixed’ in the avirulent form.

Genetic Studies with Vaccinia Virus
FRANK FENNER*®

Vaccinia virus is one of the larger animal
viruses, and differs from influenza virus in that
its genetic material is DNA and not RNA.
Thanks to Edward Jenner it has been used by
man for a longer period than any other virus,
and strains with the most varied passage his-
tories are maintained in different laboratories
all over the world. Twenty four such strains
have been intensively examined in my depart-
ment over the last two years, and considerable
differences have been found in several bio-
logical properties of different strains of
vaccinia, cowpox, and rabbitpox viruses—all
of which are very closely related immuno-
logically.

Six groups of characters were studied, two
in vitro characters — production of haemag-
glutinin and sensitivity to inactivation by
heat; and four in vivo characters—pock pro-
duction on the chorioallantoie membrane,
growth in various tyfes of tissue culture, the
type of lesion produced after intradermal
inoculation of rabbits, and virulence for mice
after intracerebral inoculation. With one or
two exceptions all strains examined were dis-
tinguishable from each other. Two pairs of
strains with a number of contrasting characters
were selected for experiments on recombination.
The pairs, and the contrasting characters are
shown in Table I,

The most extensive experiments have been
carried out with rabbitpox and dermal vaccinia.

 

* Department of Microbiology, John Curtin School
of Medical Research, Australian National Univer-
sity.

The first experiments involved the inoculation
of the chorioallantoic membrane with large
doses of the two parent strains and assay of
the new virus produced twelve hours later.
Five recombinant clones were found among the
95 clones tested.

By inoculating eggs with mixtures calculated
to produce about 50 pocks of each type it was
possible to obtain a few membranes on which
two adjacent pocks of different type overlapped.
From the overlapping areas clones of virus
were obtained by single pock isolation. About

TABLE II

Examples of the stable recombinants obtained in experiments
with rabbitpox and dermal vaccinia viruses. The abbrevia-
tions are shown in Table 1. _Underlining indicates the features
in which the strain in question differs from the parent dermal
vaccinia strain

 

 

 

 

 

j 1
Strain cam | wv | ORS HA | HR
rabbitpox parent R h = PC 0 | h

derma! vaccinia |
parent | OW 1 | N h 1
R1-1 UW 1 N 0 1
R1-3 WY] inte | int, | oF 1
RI-4 fo wl WN h h
R2-3 | Woo int, | int. | h h

 

 

*int.intermediate between the behaviour of the two

parent strains.

30 per cent. of the clones obtained showed a
grouping of characters differing from that of
each of the parents. Some clones were stable
on repeated passage and others varied some-
what. Sometimes the in vivo characters differed
from those of both parent strains, but no
‘intermediates’ were obtained in heat resistance
or haemagglutinin production. Recombination
was also demonstrated between neurovaccinia
and the white variant of cowpox.

Other lines of investigation were concerned
with mutation in the poxvirus group — the
‘white variants’ of cowpox and of some of the
neurovaccinia strains providing material for
the Luria-Delbruck fluctuation test. The pro-
cess of adaptation of dermal vaccinia strains
to rapid growth in mouse brain was also being

 

 

 

TABLE I
The biological characters of the parent strains of the pairs used for recombination experiments
Chorioallanteic Membrane Intradermal : Intracerebral |
—_—_— Inoculation | Inoculation | Haemagelutinin Heat
Strain Pock Appearance | Virus Content of Rabbits | of Mice | Production Resistance
(CAM) of Pock (VC) (BS) | (MY) | (HA) ' (HR)
rabbitpox pale haemor- high (h) indurated lesion | highly virulent nil (0) high (h)
thagic (R) with purple (h} |
centre (PC) |
dermal opaque white low () red nodule (N) | non-virulent (1) high (h) ! low (I)
vaccinia cw) \ i
neurovaccinia pale haemor- high ¢h) indurated lesion | highly virulent | high (h) | high (h)
rhagic (R} with purple ch) i |
centre (PC) | j
cowpox-white opaque white low (I) red nodule (N) | non-virulent (1) | very low (1) high (bh)
variant CW)

 

 

 

 
76

examined. In one case there appeared to be a
single step change from low to high mouse
virulence, and this was correlated with a pro-
nounced change in pock appearance. In other
cases there appeared to be a gradual increase
in virulence not clearly correlated with a
change in pock appearance.

Exchange of Character Determinants between
Strains of Plant Viruses

R. J. Best*

The exchange of character determinants
between strains of a virus was first demon-
strated for bacterial viruses, and later for
influenza virus. Some bacterial and animal
viruses have now been studied sufficiently for
genetic terms and techniques to be used with
assurance and profit. So far satisfactory
evidence that a similar phenomenon occurs in
plants has been obtained for only one virus—
the virus of tomato spotted wilt (TSW). The
complicated nature of multicellular hosts and
the fact that a plant virus lesion becomes
apparent only after several virus generations,
means that the general use of genetic terms at
this stage is unjustified and perhaps presump-
tuous. However, the evidence seems to be clear
that strains of TSW virus exchange deter-
minants when they multiply together in plants,
and that the new strains which we have
isolated from mixedly infected plants combine
some of the characters of their ‘parents’.

TSW. virus is spherical, with a diameter of
about 85 my, has a thermal death point of
46°C, and has an in vitro life of less than 12
hours in the sap expressed from infected plants.
However, by using reducing agents to keep the
redox potential of solutions below the critical
value for the virus and by working in buffered
solutions at 2°C, we can keep the virus infective
for a reasonable time. The virus is readily
transmitted by mechanical inoculation. Chemi-
cal analysis and ultra-violet absorption corres-
pond to a nucleoprotein of intermediate P
content. Although chemical and _ serological
tests are not as yet available to us as markers
for different strains, the biological properties

 

THE AUSTRALIAN JOURNAL OF SCIENCE

OCTOBER

as expressed in tissue specificity and symptoma-
tology are so definite and unequivocal that
very useful information can be obtained
through them alone. The diagnostic symptoms
by which we recognise the strains are a type
of biochemical indicator, and it is assumed
that each diagnostic character is related to a
determinant which is part of the virus particle.

Over the years a number of naturally occur-
ring strains have been isolated and charac-
terized. These breed true and have been
maintained by serial transfer at regular
intervals (about fortnightly) over a period of
years. Only those characters which are
invariably associated with a strain are used
in this work.

When two of these strains (A and E) were
inoculated simultaneously onto the same plants,
a number of new strains (as well as the
originally introduced A and E) were recovered
from the mixedly infected plants. The charac-
ters of two of these new strains are tabulated
along with those of strains A and E in Table I.

Both new strains (isolated by limit dilution
technique) differ from the originals but com-
bine some of the characters of each. Strain
‘etch’ shows a character (Etch) not shown by
either of the parents. Both new strains have
been isolated from a number of different plants
in one experiment and again in a similar
experiment repeated some months later. The
strain New ‘B’ has been isolated not only from
mixedly infected tomato plants, but also from
the systemically invaded tops of N. glutinosa
plants, a host which only one of the parents
(E) invades systemically.

The frequency with which the new strains
(especially New ‘B’) occur on the mixedly
infected plants in any one experiment and the
fact that they appear whenever the experiment
has been repeated makes it unlikely that they
are mutants.

Other specially designed tests provided
further evidence against the mutant possibility.
It is considered that the interpretation which
best fits all the facts is that the new strains
arose as a result of an exchange of deter-

* Waite Agricultural Research Institute, Univer- minants between the originally introduced
sity of Adelaide. strains.
TABLE I

Illustrating the transfer of character determinants between strains A and E of the virus of tomato spotted wilt ; symbols represent
diagnostic characters (on three hosts) of strains A and E and of new strains isolated from plants mixedly infected with strains

 

 

 

A and E
Lycopersicon esculentum Nicotiana glutinosa Nicotiana tabacum
Strain Primary Systemic Primary Systemic Primary Systemic
NIP Sy Na NIP Eq Nl ¥ Sy NIP Ni R Sy
I

A + | toe FH + = = o of] + = —
E.. a - + + oF = + (4) +
New B a + = $+ = + $= +
Etch - | + - = + + - + _ + + (+) +

 

 

 

 

 

Nl =leaf neurosis.
Na=apical neurosis.
P =pigmentation.
E =etch.

Sy systemic invasion.

Yb =yeliow blotch.

C_ =chlorosis.

(R)=ringspot at high temperatures.

 

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