Foha Microbiol. 46 (4), 309- 314 (2001) http://www, biomed, cas.

cz/mbu/folia/

Streptomyces plicatus as a Model Biocontrol Agent

E.F. ABD-AiA.At!
Seed Pathology Department. Plant Patholog);Research Institute. Agriculture Research ('enter. Giza. Eg,)pt
Received 31 July 2000
Revised version 3 January 2001

ABSTRACT. Three hundred and seventy two isolates belong-
ing to the genus Streptomyces were isolated and screened tbr
chitinase production. Streptomycesphcatus was fbund to be the
best producer. The highest chitinase production were incubated
fbr 3 d at 30 ~ on buffered culture medium (pH 8.0) containing
chJtiil plus sucrose and calcium nitrate as carbon and nitrogen
sources. S. plicatus chitmase had a highly significant inhibitor)
effect on spore germination, germ tube elongation and radial
growth of k)tsartum oaTsporum l~sp. lycopersict. Altrernana
alternata and Verttctllium albo-atrum, the causal organisms of
k'usarium wilt, stem canker and I'ertictllium wilt diseases of
tomato. Application ofS. plicatus to the root system of tomato
plants before traqsplantation markedly protected tomato plants
against the tested phytopathogenic fungi tn vtvo.

Actinomycetes in general and streptomycetes in particular are known to include several species that
inhibit growth activities of many plant-pathogenic fungi (Hardy and Sivasithamparam 1995). The proposed
mechanisms resulting in biocontrol of plant pathogenic fungi are competition for substrate (Sivan and Chet
1989), antibiosis (Lee et al. 1990) and parasitism involving the action of cell-wall lytic enzymes such as
chitinase (Elad et al. 1985; Lima et al. 1999).
The ability of streptomycetes to produce chitinases was reported by many investigators (Takegawa
et al. 1997; Saito et al. 1998; Kormanec et al. 2000). The production of chitinases by various Streptomyces
species is directly influenced by both environmental factors and nutritional requirements o f individual
strains (Zheng et al. 1994; Mahadevan and Crawford 1997).

MATERIALS AND METHODS

Test fungi and host plant. Fusarium oxysporum f.sp. lycopersici as used by Abd-Allah (1995) while
AIternaria alternata and Verticillium albo-atrum were kindly provided by Dr. K.M. Ghoneem (Tag El-Ezz
Agriculture Research Station, Dakhalia, Egypt).
The seeds of the host plant (Lycopersicon esculentum MIH,. cv. Super-Marmand) were produced by
Newman Seed Co. (USA).
Isolation o f chitinolytic streptomycetes. Rhizospheric soil samples of tomato plants were collected
from different localities of Sharkia and Ismalia Governorates (India) according to Johnson et al. (1959).
Starch-nitrate medium (Waksman 1959) was used for isolation of rhizospheric streptomycetes by the dilut-
ion plate agar method (Johnson et al. 1959); starch was supplemented with chitin in equivalent amounts. The
plates were incubated for 10 d at 28 + 2 ~ The developed isolates were streaked on agar plates to obtain
single colony and stored in slope agar tubes at 4 ~ until used.
Screening f o r quantitative chitinase production by isolated streptomycetes. Three hundred and
seventy two isolates were grown (about 108 spores used for inoculation) on chitin-nitrate broth medium
(50 mL culture medium in 250-mL Erlenmeyer flasks; pH 7.2) at 28 + 2 ~ on a rotary shaker (1.7 Hz) for
7 d. At the end of incubation, the cultures were centrifuged (10 000 g, 10 rain) and supernatants were used as
enzyme source. The precipitate was washed with 0.5 mol/L HCI (for dissolving the residual CaCO3) and
dried at 80 ~ until constant mass; growth was expressed as g of dry mass per L culture medium.
Enzyme assay. Chitinase (l,4-[3-poly-N-acetyl-o-glucoaminidase, EC 3.2.1.14) was assayed by fol-
lowing the release of N-acetyl-o-glucosamine (Reissig et al. 1959) from colloidal chitin (prepared according
to Campbell and Williams 1951). A reaction mixture containing I mL of crude chitinase (culture filtrate),
1 mL of 0.1 mol/L citrate-phosphate buffer (pH 5.1) and 1.6 mg colloidal chitin was incubated (38 ~
2 h): the reaction was stopped by boiling. One unit (U) ofchitinase activity was equal to 1 j.tmol of N-acetyl-
o-glucosamine per I h. Soluble chitinase activity was expressed as 1 U/mL of culture filtrate. Soluble
protein concentration was measured according to Lowry with bovine serum albumin as a standard.
Taxonomic characteristics o f the selected Streptomyces isolate. The most active Streptomyces iso-
late (producing the highest amounts of extracellular chitinase) was characterized according to International
Streptomyces Project (Shirling and Gottlieb 1966) and Bergey's Manual o f Systematic Bacteriology (Stan-
ley and Felsche 1989). Spore-chain morphology was observed using the microscope film camera Karl-Zeiss-
310 [" F ABD-AI.IAH Vol 4()

,lena (Germany). Direct mounting of spores on colloidal films was examined using transmission electron
microscope (100 CX, Jeol, Japan: collaboration with the Facul O, oJScience, Zagazig University, Egypt) by
the spore-print technique (Tresner et al. 1961).
Environmental and nutritional conditions Jot growth and chitinase production Starch-nitrate broth
medium (Waksman 1959) was used during all studies of environmental and nutritional conditions affecting
chitinase production by S plicatus. Citrate--phosphate buffer was used for pH adjustment of the basal me-
dium, supplemented with different carbon sources (added to chitin in 50 %, W/W) and different amounts of
nitrogen sources.
Ammonium sulJate precipitation ofchitinase. The cell-free culture fltrate was brought to 95 % satu-
ration with (NH4)2SO4 at 0 ~ for 2 h according to Mauch et al. (1988a). The precipitate was collected by
centrifugation (2000 g, 20 min), resuspended in 50 mmol/L sodium acetate buffer (pH 5.0) and dialyzed
against water at 4 ~ for 14 h and then against 50 mmol/L sodium acetate buffer for 2 h. The dialyzed solut-
ion was used as crude enzyme preparation for antifungal assay.
Antifimgal activiO, on spore germination as well as germ-tube length of the plant pathogenic fungi
was estimated according to Roberts and Selitrennikoff (1988).
Effect ofchitinase onfungal growth Chitinase was passed through a sterile Millipore nylon mem-
brane filter (0.45 pro) for sterilization before its application as fungistatic agent according to EI-Abyad et al.
(1983).
Biological control of pathogenic.fungi of tomato plants in vivo. A glasshouse experiment was car-
ried out in the Botany Department. Faculty of Science, Zagazig University (Zagazig, Egypt). The sandy clay-
loam soil collected from a tomato field had the following properties: organic carbon 0.52 %; total soluble
salts 0.30 %; moisture holding capacity 14.8 %; pH 7.5. The soil was dried and mixed with fine sand at the
ratio of 2 : 1 (W/W) and autoclaved (3 h, 121 ~ Cooled soil was divided into plastic pots (400 mm dia-
meter, 5 kg capacity).
Treatments. Ten pots were used for each treatment, each containing three tomato seedlings. The
tomato seeds were surface-sterilized with 1% (V/V) sodium hypochlorite for 10 rain and thoroughly washed
with sterile distilled water before germination. Similar healthy seedlings (150 mm in length) resulting from
sown tomato seeds were used in control experiment. The roots of tomato seedlings were soaked in spore sus-
pension ofS. plicatus (200/nL, i.e. 2 x 108 per mL) in 1% (W/V) carboxymethyl cellulose as sticker for ] h.
The soil was infested by the pathogens before the sowing at the rate of 0.2 g/kg soil (Rothrock and Gottlieb
1984); control pots were used for each treatment.
Disease index was evaluated as the percent of plants showing identical symptoms of wilt (Fusarium
oxysporum f.sp. lycopersici and Verticillium albo-atrum) and stem canker (Alternaria alternata).
Statistical analysis For each experiment, the data were statistically analyzed using analysis of vari-
ance for a completely randomized design. Treatment means were compared using the protected least signi-
ficant difference (LSD) values according to Daniel (1987).

RESULTS AND DISCUSSION

The most active isolate is a member of the gray series from the rhizosphere of Peto-86 tomato
cultivar grown in Ismalia Governorate (Egypt).
Diagnostic characteristics and identification ()./'the isolate. Spore chain morphology. Sporophores
are spiral (Fig. IA); spore surface is smooth with about 50 spores per chain (Fig. 1B).
Color. Aerial mycelium is gray on inorganic salts-starch agar, oat meal agar and Czapek's solution
agar; on glycerol-asparagine agar, malt-yeast extract agar and starch-nitrate agar it is light gray, and on
glucose-nitrate agar, glycerol-nitrate agar, potato agar and fish meal-agar reddish-gray. The color of the
substrate was yellowish-brown on glycerol-asparagine agar and malt-yeast extract agar, pale yellow on
Czapek's solution agar, and grayish yellow on the other media. The substrate mycelium pigment was not
a pH indicator when tested with 50 mmol/L NaOH or 50 mmol/L HCI. Melanoid pigments were not formed
on tyrosine agar, peptone -yeast-iron agar and tyrosine broth.
Carbon utilization Good growth was observed on the following carbon sources: D-glucose, L-arabin-
ose, D-xylose, L-rhamnose, D-fructose, myo-inositot and D-mannitol. No growth was observed on raffinose or
sucrose.

The isolate showed sodium chloride tolerance up to a concentration of 10 % (W/k).

_Antibiotic production. A diffusible antimicrobial substance inhibiting the growth of Bacillus mega-
terium, B. subtilis, Staphylococcus aureus, Micrococcus lutea and E. coli was produced; it did not affect the
growth of Pseudomonas aeruginosa and Penicillium chrysogenum.
2001 CLONING AND NUCLEOTIDE SEQUENCE OF DNA PI.ASMID FROM X. denclrorhous 283

DISCUSSION

Linear DNA plasmids are common among filamentous fungi and plants in which they are gen-
erally associated with mitochondria. In contrast, the linear DNA plasmids of yeasts appear to be of
cytoplasmatic origin (Fukuhara I995), we did not yet investigate the location of the elements corre-
sponding to the bands pPhl and pPh2 in X. dendrorhous. The pPhl group could represent three differ-
ents plasmids of 6.9 (pPhll), 5.7 (pPhl2) and 4.7 kb (pPhl3), apparently present in a high copy num-
ber. On the other hand, the pPh2 group, represented by two bands of 3.6 (pPh21) and 3.0 kb (pPh22),
if they turn out not to be derived from those of the pPhl group, are present in a low copy number.
Southern blot hybridization of the oligonucleotide primer from the pDK1-D sequence con-
firmed that pPhl3 is an extrachromosomal molecule and that there was no integration of it into the
genome of X. dendrorhous. The plot indicates if all the molecules represented by the groups pPhl and
pPh2 are really different, some degree of similarity exits at least in the region corresponding to the
oligonucleotide sequence used as a probe.
pPhl3 appears to be 4.7 kb long, also the insert into pKD1 seems to be 4.7 kb as measured in
conventional agarose-gel electrophoresis. Considering that the entire sequenced insert from pKD1
comprises 4077 bp and that the mobility is shifted from 4.7 to 4.0 kb when run in a denaturing gel as
well as the putative secondary structures that we describe, we conclude that the size of pPhl3 deter-
mined by agarose electrophoresis could also be biased and that the insert of pKD1 could represent
almost the entire pPhl3. The complete sequence of pPhl3 may be slightly larger since the precise
nature of the termini is not yet known.
Various linear genomes have inverted terminal repeats, for example, pGKL1 and pGKL2 from
Kluyveromyces lactis (Sor et al. r983). Some of those have covalently attached proteins to the 5'-ter-
mini, like adenovirus DNA (Carusi t977) and also pGKL1 and 2 from K. lactis (Hishinuma et al. t984).
Those proteins make the linear DNA inaccessible for phosphorylation by polynucleotide kinase even
after treatment with bacterial alkaline phosphatase. But also cytoplasmic plasmids not blocked at the
5'-end were found, like the linear mtDNA plasmid from the yeast Hansenula mrakii (W~solowski and
Fukuhara ,98 0. The linear plasmids pLPO1 and pLPO2 present in the mitochondria of the basi-
diomycete Pleurotus ostreatus (Yui et al. x988) are also blocked at their 5'-ends by an associated pro-
tein. Phenol extraction results in the disappearance of the protein-associated plasmid DNAs from the
aqueous phase, which can be avoided by prior treatment with proteinase K. In our case, the phenol
extraction in the absence of proteinase K treatment did not decrease the yield of pPhl and pPh2; this
observation indicates that if there is some related protein at the 5'-end, the association is not very tight.
We also did not find internal palindromic sequences within the known terminal sequences. The
presence of those internal palindromes has been proposed by Cavalier-Smith (1974) to solve the prob-
lem of filling the 5'-end in the synthesis of linear DNA. It has been reported previously that the termi-
nal repetitions may contain several short dispersed repeats. In our case we cannot be sure that the
extremes of either pPhl or pPh2 are represented on pDK1. But we observed many terminal repeti-
tions, 35 bp long, inverted at each end. This differs from the quite universal short tandem repeats, pre-
sent in almost every telomere structure of linear chromosomes. Before we determine whether pDK1
includes the total extremes of pPhl3, as well as prove the presence or absence of blocking proteins, we
cannot determine which class of linear plasmid they resemble.
The presence of the 32-bp inverted repeat sequence near the end of the ORF P2 resembles the
termination hairpin in prokaryotic cells (Birnstiel 2985). The ll-bp sequence (6GAGTC 6AA r ) in this
putative hairpin, and also after and before the starting point of the ORF P2, could play some regulatory
role in the expresion of ORF P2. There is another 15-bp (GGr GGT CTT ATe AGA) segment present
twice before the ORF P1 and one of them comprises the total loop of a putative hairpin loop structure
(1757-1799). Whether these sequences have any significance in the expression of the putative proteins
coded by the ORF, remains to be elucidated.
By means of computer analysis of the sequence data we did not find similarity within any pro-
tein and translated ORF from the database sequences. However, the most interesting finding is a 22-bp
(AGAAGAAGAGGAGGAAAAGAAA) sequence; this was found with an expected value of 4 x 10.-4 in
the ZDS1 gene of Saccharomyces cereuisiae. This gene, also called NRC1, CES1 or R T G 2 s l , stabilizes
short linear centromeric plasmids when present in multiple copies per cell (Roy and Runge 1999).
 312 E.F. ABD-ALLAH Vol 46

( E l a d et al. 1985) and Trichoderma harzianum ( S i v a n a n d C h e t 1989) w e r e u n a b l e to d e g r a d e live m y c e l i u m

o f F o.~vsporum. T h e d i f f e r e n c e in the r e s p o n s e o f t h e t e s t e d fungi to c h i t i n a s e a c t i v i t y w a s c o n s i s t e n t w i t h
o t h e r r e p o r t s ( S c h l u m b a u m et al. 1986; M a u c h et al. 1988b) and w a s d e p e n d e n t o n chitin c o n c e n t r a t i o n in
cell w a l l s o f the t e s t e d f u n g i ( B a r t n i c k i - G a r c i a 1968).

Table I. Growth (g/L) and chitinase production (U/mL) by S pficatus

Condition Value and/or effector Mycelial growth Final pH a Chitinolytic activity

Incubation. d 1 0.37 6.3 12.3

2 0.82 6.8 26.4
3 1.29 7.4 38.0
4 2.37 7.9 32.8
5 1.84 8.4 26.1

LSD at 0.05 0.49 0.88 33.7

0.01 0.71 1.26 48.0

Temperature, ~ I0 0.72 6.8 15.1

20 1.00 7.3 27.6
30 1.31 7.6 38.6
40 5.63 7.2 36.8
5O 0 7O 0

LSD at 0 05 0.08 33 18.9

0.01 0.12 47 26.9

pH 4.0 0.30 - 8.5

5.0 0.78 - 12.8
6.0 1.30 - 19.5
7.0 1.57 - 35.2
8.0 2.20 47.0
9.0 1.81 31.6

LSD at 0.05 0.08 0.84

0.01 0.12 1.18

Carbon sourceb Chitin (control) 2.22 - 46.2

+ glucose 0.99 - 0
+ mannose 3.16 - 61.1
+ sucrose 4.49 - 70.0
+ mannitol 1.69 - 12.4
+ N-acetylglucosamine I. 17 - 6.7
+ starch 1.49 - 4.1

[.SD at 0.05 0.15 - 16 I

001 0.20 - 22.3

Nitrogen sourcec KNO 3 (control) 2.0 2.25 - 46.7

Ca(NO3) 2 2.3 2.82 - 68.4
(NH4)2SO 4 1.3 1.07 - 9.3
(NH4)2HPO 4 1.3 2.04 3.5
NH4CI 1.0 1.50 - 11.9
CO(NH2) 2 0.6 2.37 - 33.4

LSD at 0.05 0.12 - 0.87

0.01 0.17 - 1.22

alnitial pH 7.0. bAdditional carbon sources supplied in equimolar amounts with chitin. Cg/L

A p p l i c a t i o n o f S . plicatus t o m a t o r o o t s b e f o r e p l a n t a t i o n s i g n i f i c a n t l y r e d u c e d t h e d i s e a s e i n c i d e n c e
( T a b l e III). C h i t i n o l y t i c a c t i v i t y is c o n s i d e r e d an i m p o r t a n t f a c t o r in p l a n t p r o t e c t i o n and in b i o c o n t r o l o f
s o i l - b o r n e p l a n t p a t h o g e n i c f u n g i (e.g., K a p a t et al. 1998). T h e i n h i b i t o r y e f f e c t o f c h i t i n a s e f r o m o u r Strep-
tomyces strain o n s p o r e g e r m i n a t i o n , g e r m t u b e e l o n g a t i o n a n d radial g r o w t h o f t e s t p a t h o g e n i c f u n g i c o n -
f i r m s t h e role o f c h i t i n a s e in p l a n t d i s e a s e control.
T h e ability to p r o t e c t t o m a t o p l a n t s b y a p p l i c a t i o n o f c h i t i n o l y t i c S. plicatus o p e n s the w a y for t h e
d e v e l o p m e n t o f c h i t i n a s e as an a l t e r n a t i v e to d i r e c t c o n t r o l o f s o i l - b o r n e p l a n t p a t h o g e n i c fungi.
2001 MOI)t'~I. B I O C O N T R O I . AGI!NI - S phcums 313

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:314 E F ABD-AI, t.AF[ Vol 46

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