Physiological and Molecular Plant Pathology (1987) 30,199-206

Correlation between albicidin production and chlorosis

induction by Xanthomonas a/bi/ineans, the sugarcane leaf
scald pathogen
ROBERT G . BIRCHt
Bureau of Sugar Experiment Stations, P .O . Box 86, Indooroopilly, Q ; 4068, Australia

and SURESH S . PATIL

Department of Plant Pathology, University of Hawaii, Honolulu, Hawaii 96822, U.S.A .

(Acceptedfor publication August 1986)

Chlorosis of emerging leaves of sugarcane plants invaded by Xanthomonas albilineans results from
blocked chloroplast differentiation, apparently caused by a phytotoxin . Chlorosis inducing isolates
of X. albilineans produce a family of antimicrobial compounds, including albicidin which inhibits
prokaryote DNA replication . The objective of this study was to determine whether albicidin, or a
related compound produced in diseased plants, causes chlorosis of uninvaded tissues characteristic
of sugarcane leaf scald disease . Spontaneous loss of albicidin production occurs at a high rate but
does not involve extrachromosomal genetic elements . Results with mutants and revertants in
albicidin production show a close correlation between albicidin production and ability to cause
chlorosis, strongly suggesting that the chlorosis inducing toxin is biosynthetically closely related to
albicidin. Plasmid pJB4JI is transmitted at high frequency from Escherichia coli to X. albilineans,
where it is stably maintained as a conjugative plasmid, providing a basis for further genetic analysis
of X . albilineans using well-characterized PI group plasmids .

INTRODUCTION

Xanthomonas albilineans is a xylem-invading bacterium causing chlorosis of emerging

leaves of sugarcane (inter-specific hybrids of Saccharum spp) and artificially inoculated
sweet corn (Zea mays L . van saccharata Bailey) . Chlorosis results from blocked chloroplast
differentiation in parenchyma surrounding invaded xylem vessels, and in uninvaded
white leaves emerging after invasion of sugarcane stalks by the pathogen . Although the
visible and ultrastructural symptoms suggest involvement of a chlorosis-inducing phyto-
toxin, direct assays have failed to demonstrate production of such a toxin by the patho-
gen in culture [3] . However, chlorosis inducing isolates of X. albilineans produce a
mixture of related antimicrobial compounds in culture . The major, purified anti-
microbial compound (albicidin) is bactericidal to Escherichia coli, causing a rapid and
complete block to DNA synthesis, followed by partial inhibition of RNA and protein
synthesis [4] . Because chloroplasts employ prokaryote mechanisms of DNA replication,
transcription and translation it seemed possible that albicidin, or a structural analogue

tTo whom all correspondence should be addressed .

0885-5765/87/020199+08 $03 .00/0 © 1987 Academic Press Inc. (London) Limited

200 R . G . Birch and S . S . Patil
produced in diseased plants, might be involved in the blocked chloroplast differentiation
which characterizes sugarcane leaf scald chlorosis . To examine this possibility we have
tested the ability of mutants and revertants in microbial-inhibitor production to cause
chlorosis in inoculated plants . The results support the hypothesis that a compound
biosynthetically closely related to albicidin causes chlorosis in sugarcane leaf scald
disease .

MATERIALS AND METHODS

Bacterial strains and microbial assay

Isolates of X. albilineans were obtained as described by Persley [21] from stalk and leaf
tissues of sugarcane varieties H58-8029, H60-6314, BN68-8654 and Q63 showing
characteristic symptoms of leaf scald disease . Original isolates used in this study are listed
in Table 1 .
To test for production of antimicrobial compounds cells of each strain were spotted
onto plates of sucrose peptone (SP) agar using sterile toothpicks, and incubated at 28 ° C
for 5 days . Plates were then overlaid with E . coil UHP1 or UQM 70 as described
previously [4] and examined for inhibition zones after 12 h at 37 °C .
To determine whether inhibition of E. coil by original isolates of X. albilineans used in
this study was due solely to albicidins, these strains were tested for inhibition of E. coil
mutants selected for resistance to albicidin . Mutants of E . coli show no cross-resistance
between albicidins and other known families ofantibiotics [4] ; (R . G . Birch, unpublished
data) .

Production of X . albilineans mutants

Additional non-inhibitory mutants, and revertants with restored inhibitor production,
were sought to test an apparent correlation in X. albilineans strains between production of
albicidins and ability to cause chlorosis in infected sugarcane . Several approaches were
used .
(1) Dilution plates from log-phase SP broth cultures, and plates spotted with
colonies recovered after five weeks growth on SP agar, were overlaid with E. coli to detect
spontaneous non-inhibitory mutants .
(2) Nitrosoguanidine (NG), ethyl methane sulfonate (EMS), 2-aminopurine
(2-AP) and ultraviolet (UV) mutagenesis were carried out as described by Miller [18] .
(3) Transposon-insertion mutagenesis was initially attempted by introduction of
"suicidal" plasmid pJB4JI carrying transposon Tn5 from E. coli 1830 [1] into X.
albilineans LS2 by filter matings followed by plating on minimal medium [4] plus
50 µg ml - ' kanamycin to select transconjugant X. albilineans . We subsequently used
filter and patch matings to transfer pSUP5011 and pSUP1021 from E. coli S 1 7-1 [22] for
Tn5 mutagenesis of X. albilineans LS 116 .

Plant inoculations and re-isolations

Non-inhibitory clones (assumed to be mutants) and inhibitor-producing revertants were
rechecked for inhibitor production after serial single colony transfers, then used to inocu-
late sugarcane and sweet corn plants as described previously [3] . Fifteen days after
inoculation of sugarcane plants, 1-cm strips were removed 4 cm below the cut end of the
Correlation between albicidin production and chlorosis induction 201
TABLE I
Symptom production by original isolates of Xanthomonas albilineans and mutants altered in inhibitor
production

White pencil lines in inoculated

Inhibition of
Strain Origin E. coli Sweet corn Sugarcane

LS1
LS2 • + +
LS3 • + +
LS4 • + +
Original isolates
LS5 • + +
LS6 • + +
LS106 • + +
LS1 16 • + +
LS7 Spontaneous ex LS2
LS8 UV ex LS2
LS9 UV ex LS2
LS I O 5 wk culture ex LS2 -
LS I l UV ex LS2
LS 12 UV ex LS2 -
LS 13 UV ex LS2
LS 14 UV ex LS4 -
LS 15 -
LS16 -
LS17 Spontaneous ex LS2
LS18 -
LS19
LS20 NG ex LS2 ++b
LS21 Spontaneous ex LS2
LS24
NG ex LS2
LS25 } -
LS107 UV ex LSI06
LS10
NG exLS106
LSI18 } -
LS127
Spontaneous ex LSI16
LS128
LS129
EMS exLSI16
LS130
LS 131
Tn5 ex LS 116
LS132 ~ - - -

'Apparent "leaky" mutants, producing small inhibition zones at longer preincubation times.
'Mutant selected for four-fold higher production of albicidin in culture .
`Strains caused chlorosis of emerging leaves after invading stalks, see text .

second youngest cut leaf, surface-sterilized with 70% ethanol, ground in a mortar with
2 ml of sterile water, allowed to stand 2 h at 24 ° C, then dilutions were plated on dupli-
cate SP agar plates . After 9 days of incubation at 28 ° C recovered colonies were counted
then overlaid with E. coli to check for inhibitor production .

RESULTS
In our initial test, five chlorosis-inducing isolates of X. albilineans were observed to
produce a microbial inhibitor, whereas an exceptional non-chlorosis-inducing isolate
failed to inhibit E. colt (Table 1, LS 1 to LS6) . These isolates were typical of X. albilineans
202 R . G . Birch and S . S . Patil
in all bacteriological characteristics [2], and LS I is considered to be a naturally
occurring chlorosis - mutant .
Inhibition of E. colt by original isolates of X. albilineans under the assay conditions
described was due solely to albicidin or related compounds, as indicated by complete
absence of inhibition of albicidin-resistant mutants of E. colt .
The frequency of non-inhibitory colonies on plating diluted log-phase cultures of
LS2 and LS4 was 1 x 10 -3 to 6 x 10 -3 , and sometimes increased greatly in old cultures .
In one trial all colonies of LS2 recovered after 5 weeks of growth on SPA plates proved
non-inhibitory whereas recovered LS4 colonies continued to produce the inhibitor . After
UV irradiation of cells from log-phase liquid cultures to 99 . 5% kill, or NG treatment to
50% kill, frequency of non-inhibitory colonies among survivors increased to I X 10 -2 to
3 x 10 -2 and 3 x 10 -2 to 10 -1 , respectively . Properties of original isolates and indepen-
dently derived non-inhibitory mutants are shown in Table 1 .
All strains selected for loss of inhibition of E. coli failed to cause white pencil lines in
inoculated sweet corn and sugarcane, in contrast to inhibitor-producing parent strains
and a mutant with increased inhibitor production, which continued to cause chlorosis in
inoculated plants . Several non-inhibitory strains were recovered from stems ofsymptom-
less sugarcane plants four months (LS1, LS9) to two years (LS19, LS24) after inocu-
lation, and confirmed not to inhibit E. colt . However, three of 19 tested non-inhibitory
strains caused chlorosis of emerging leaves after a prolonged latent period (4-6 months)
in systemically invaded sugarcane . These strains (LS15, LS 18, LS21) were reisolated
from stalk tissue of plants with emerging chlorotic leaves and side-shoots, and retested for
inhibition of E . colt . None of the several thousand colonies recovered from stalk segments
at various heights, or from the bases of chlorotic side shoots, caused the clear inhibition
zones typical of albicidin producing parent strains . However, many reisolated colonies
caused very turbid inhibition zones in the overlayer . Albicidins produced in culture by
X. albilineans LS2 cause clear zones of inhibition of E. colt, with zone diameter pro-
portional to antibiotic concentration [4] . The compounds produced by many reisolated
colonies of LS 15, LS 18 and LS21 which caused very turbid inhibition zones were not
isolated or characterized relative to albicidin .
Strains LS 1, LS7, LS8, LS9, LS 10, LS 12, LS 13, LS 15, LS 16, LS 17, LS 18, LS21,
LS24, LS 107, LS 110 and LS 118 were screened for spontaneous or NG-induced rever-
tants to inhibitor production . Strains LS 129 and LS 130 were screened for 2-AP induced
revertants to inhibitor production . A minimum of 10 3 colonies from log-phase cultures of
each strain was screened, both before and after NG or 2-AP mutagenesis . Strains LS 1
and LS13 yielded spontaneous revertants to inhibitor production at a frequency of
4 x 10 -3 to 6 x 10 -3 per colony plated, increasing to 3 x 10 -2 to 9 x 10 -2 after
mutagenesis . No back-mutants were detected from other strains . Although LS1 and
LS13 appear to be leaky mutants, producing small inhibition zones at longer pre-
incubation times, no white pencil lines or systemic chlorosis resulted in sweet corn and
sugarcane plants inoculated with either strain in repeated trials . Three spontaneous
inhibitor-producing revertants selected from these strains caused white pencil lines in
both hosts ; and of 40 NG-induced back-mutants selected for inhibition of E . coli, 93%
and 44% caused white pencil lines in sweet corn and sugarcane, respectively .
Table 2 shows populations recovered from sugarcane leaves inoculated with a range
of X. albilineans strains varying in inhibitor production and chlorosis induction . The
Correlation between albicidin production and chlorosis induction 203
TABLE 2
Symptom production and multiplication in inoculated sugarcane by original isolates of Xanthomonas
albilineans, and mutants and revertants altered in inhibitor production

Population (bacteria cm -2)'

Inhibition of White pencil
Strain Origin E. coli lines Expt. 1 Expt. 2

LS I Original isolate - - 2 x 10' NT

LS2 Original isolate + + 10 6 5 x 10 5
LS4 Original isolate + + 10' 3 x 10 5
LS7 Spontaneous LS2 - 2 x 10 5 NT
LS9 UV ex LS2 - - 10 6 NT
LS11 UV ex LS2 - 10 5 NT
LS12 UV ex LS2 - 2 x 10' NT
LS13 UV ex LS2 - 3 x 10 5 2 x 10 3
LS14 UV ex LS4 - - NT 2 x 10 6
LS 15 Spontaneous ex LS2 - - NT 4 x 10 3
LS21 Spontaneous ex LS2 NT 8 X 10 s
LS30 NG ex LS13 + - NT 2 x 10 5
LS32 NG ex LS l3 + + NT 5 x 10 6
LS43 NG ex LS13 + NT 10 2
LS47 NG ex LS I + + NT 10'
LS57 NG ex LS I + NT 10 5
LS59 NG ex LS 1 + NT 6 x 10 3
LS61 NG ex LS I + - NT S 10 2
LS62 NG ex LS I + - NT 5 x 104
LS81 Spontaneous ex LSI + + NT 10°

'Recovery of pathogen 4-5 cm below cut end of leaf, 15 days after inoculation ; threshold for
detection= 10 2 bacteria cm -2 .
NT = Not tested .

range in recovery, from 2 x 10 3 to 2 x 10 7 bacteria cm -2 leaf tissue, probably arises in

part from invasion by the xylem-inhabiting pathogen of a low but variable number of
vascular bundles in each inoculated leaf. This non-uniform invasion pattern precludes
the precise quantitative comparisons possible with mesophyll invading pathogens, but
several important relationships are apparent : (1) the highest populations detected for
inhibitory and non-inhibitory strains were equal, and (2) although failure of several
inhibitor producing revertants to cause white pencil lines may reflect inability after
several cycles of mutagenesis to multiply in sugarcane leaves (LS43, LS61), others
reached substantial populations without causing chlorosis (LS30, LS57, LS59, LS62) .
Reisolates tested for inhibition of E . colt conformed with the inoculum in all cases except
for one of 165 reisolated colonies of LS13 which had regained inhibitor production,
presumably by spontaneous back-mutation .
Because of the observed high rate of spontaneous loss of inhibitor production, several
techniques were applied to test for extrachromosomal control of inhibitor biosynthesis .
Growth of LS2 and LS4 with sublethal concentrations of acridine orange (0 . 5 tg ml -1 ) ,
ethidium bromide (0 . 1 .tg ml -1 ) or elevated temperature (37 ° C), commonly used
plasmid curing treatments [11] did not increase the frequency ofnon-inhibitory variants .
No plasmids were detected in strains LS1, LS2 or LS4 using the technique of Portnoy &
White [12] which readily demonstrated all plasmids in Agrobacterium tumefaciens and
204 R . G . Birch and S . S . Patil
Pseudomonas syringae pv . phaseolicola controls . Cross-streaking of inhibitory and non-
inhibitory strains followed by UV treatment [9], temperature shock to agar overlay
cultures [17] and negative stain electron microscopy on mitomycin C lysates [7]
revealed no bacteriocins or temperate phages in strains LS1, LS2 and LS4 . Because
extrachromosomal elements appear to be lacking, the high rate of spontaneous variation
in inhibitor production presumably arises from instability in chromosomal genes
involved in albicidin biosynthesis [15] .
The frequency of spontaneous kanamycin resistant mutants in cultures of LS2 was
below the detection limit of 10 -9 per cell plated . Frequencies of transfer of kanamycin
resistance from E. coli 1830 (pJB4JI) to LS2 during 2 and 14 h filter matings were
3 x 10 -4 and 5 x 10 -2 per recipient plated, respectively . Of 108 transconjugants tested,
106 were resistant to kanamycin (50 .tgml -1 ), streptomycin (50 tgm1 -1 ) and genta-
mycin (5 gg ml -1 ), and the remainder were resistant to kanamycin and streptomycin
but sensitive to gentamycin . Two transconjugant clones of each type were examined for
plasmids as described above, and found to have gained a plasmid that co-migrated in
agarose gel electrophoresis with pJB4JI from the E. colt 1830 parent . To determine
whether pJB4JI could act as a conjugative plasmid in X. albilineans, four independent
streptomycin resistant mutants, and four rifampicin resistant mutants were selected from
LS2 using a gradient plate technique [12] . In spot matings on plates selecting against
both parents [18], all four plasmid-bearing clones transferred kanamycin resistance to
rifampicin resistant recipients, whereas no progeny developed in crosses between the
spontaneous streptomycin resistant and rifampicin resistant clones . Thus pJB4JI was
stably maintained as a conjugative plasmid in X . albilineans, and proved unsuitable as a
vector for transposon mutagenesis . Recent reports indicate that the Mu-based suicide
vector pJB4JI also survives in Rhodopseudomonas [14] Rhizobium and Agrobacterium spp . due
to high frequency deletions in Mu DNA [8] . Alternative suicide vectors have therefore
been developed and we found pSUP5011 and pSUP1021 suitable for transposon muta-
genesis of X. albilineans, yielding Tn5-insertion mutants at a frequency of 10 - ' to 10 -$
per recipient plated .

DISCUSSION
The close correlation between production of albicidin or related inhibitors and chlorosis
induction in invaded plants strongly suggests that the chlorosis inducing toxin (or toxin
mixture) is biosynthetically closely related to albicidin .
As a non-peptide antibiotic containing several aromatic rings and approximately 38
carbon atoms [4] albicidin must necessarily arise by a stepwise biosynthesis, with each
step catalysed by a specific gene product . Moreover, albicidin appears to be one of a
mixture of chemically related inhibitors produced in culture by X. albilineans, as all
components are chromatographically similar [4] and E . coli mutants selected for
resistance to pure albicidin are resistant to the mixture of inhibitors (R . G . Birch,
unpublished observations) . This is typical of secondary metabolite biosynthesis, reflect-
ing relative insensitivity of the involved enzymes to variations in the substituent functions
of percursors . The consequence of this loose substrate specificity is a biosynthetic "grid"
rather than a linear pathway, so that in mutants lacking one pathway enzyme, other
enzymes normally functioning later in the sequence may still operate to produce a shunt
metabolite [13] . For example, 27 tetracycline-like compounds have been isolated from a
Correlation between albicidin production and chlorosis induction 205
biosynthetic grid with 11 enzymes [23] . The proportion of each component in a mixture
depends on both genetic and environmental influences [16] . These factors, exemplified
by the tetracycline and aminocyclitol antibiotics [20,23] prevent complete elucidation of
secondary metabolite biosynthetic pathways by mutant analysis alone .
However, the existence of mutants which cause systemic chlorosis in sugarcane with-
out causing clear inhibition zones against E . coli (LS15, LS18, LS21) and of mutants
which inhibit E . coli and multiply in sugarcane without causing chlorosis (LS30, LS57,
LS59, LS62) indicates that albicidin itself cannot be the sole phytotoxin, or a necessary
component of a phytotoxic mixture, unless these are regulatory mutants repressed for
albicidin production only in culture or only in sugarcane . By analogy with the tetra-
cyclines [10] one interpretation of results with albicidin mutants is that because of the
differential permeability of E . coli and sugarcane cells, some members of the albicidin
family may penetrate to the target site in only one of these cell types .
Isolation of the effective vivotoxin may be difficult for lack ofa suitable bioassay if this
component is a less effective antibacterial agent than albicidin, as seems likely from the
mutant study . However, several approaches are available to further test the hypothesis .
Genetic analysis might be used to determine the number and arrangement of genes
common to albicidin synthesis and phytotoxin synthesis . Genetic studies in X. albilineans
have not previously been reported, but the high transmission frequency of pJB4JI from
E. coli, and its stable maintenance in, and conjugative transfer between, X. albilineans
strains, suggest the possibility for genetic mapping in this species by chromosome
mobilization and recombinant DNA techniques using well-characterized P1 group
plasmids [6] . The capacity to generate Tn5-Mob insertions in albicidin biosynthesis
genes using pSUP5011 should also be useful in this respect . As an alternative to the
genetic approach, we have tested production of albicidin by X. albilineans in infected
plants, and effects of albicidin on isolated plastids, tissue cultures and intact sugarcane
plants . The results, reported in an accompanying paper, support and extend those from
the study of mutants in albicidin production [5] .
In contrast to the bean halo-blight system [19] non-chlorosis-inducing strains of X .
albilineans are able to invade sugarcane systemically . However sugarcane plants inocu-
lated with non-chlorosis-inducing strains have not developed any acute symptoms of leaf
scald disease in 6-12 months under glasshouse conditions . To determine the role of the
toxin in overall disease development we have commenced field trials comparing disease
severity in leaf-scald susceptible sugarcane cultivars inoculated with wild type X .
albilineans and non-chlorosis-inducing mutants . If such mutants fail to induce severe
disease, attempts to select toxin-resistant variants of agronomically superior but
leaf-scald susceptible sugarcane cultivars would be warranted .

We gratefully acknowledge partial financial support for this research under Hatch
Project HAW00747-H and ARGS Grant D284/16258 . We are grateful to F . J . McKay
for capable technical assistance during part of this research .

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