Microb Ecot(1989) 18:175-186

MICROBIAL ECOLOGY 9 Springer-VerlagNew York Inc. 1989

The Bacterial Microflora of Witloof Chicory (Cichorium intybus L. var. foliosum Hegi) Leaves M. F. Van Outryve,* F. Gosselr,** and J. Swings Plant Genetic Systems,Plateaustraat22, B-9000 Gent, Belgium Abstract. The bacterial flora on the heads of four different witloofchicory varieties was examined. The 590 isolates were characterized by their SDSPAGE protein profilesi they revealed 149 different protein fingerprint types. The fluorescent Pseudomonas fingerprint type CH001 was abundantly found on all heads examined. Fourteen other fingerprint types occurred in high densities more than twice. Among these, the following were identified: fluorescent Pseudomonas, nonfluorescent P s e u d o m o n a s sp., Erwinia herbicola, Erwinia sp., and Flavobacterium sp. The majority of the fingerprint types (90%) was found only once. It was also our objective to isolate bacteria applicable in the biological control of chicory phytopathogens. Isolates of all fingerprint types were tested for in vitro antagonistic activity and for possible deleterious effect on plant growth. Fluorescent P s e u d o m o n a s and Serratia liquefaciens isolates were antagonistic against fungi. Among the 161 fluorescent P s e u d o m o n a s strains, five were able to produce disease symptoms on chicory leaves upon inoculationJComparison of the results of this study with those obtained in two previous analyses revealed that the leaf microflora showed some similarities with the bacterial flora of chicory roots. The chicory seed microflora differed from that ofbot'h leaves and roots.

Introduction

An inventory of the bacterial population on plant surfaces allows not orfly ~i better understanding but also a more efficient exploitation of the microbe--plant interactions Ihat may directly or indirectly i~nfluence fhe plant. The classifications of high numbers of isolates from complex environments still poses serious problems. Numerical analysis of phenotypic features has been applied for the identification of large numbers of phyltoplane bacteria on L o l i u m p e r e n n e [3], Olea europaea [14], and several other plants [15]. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of-bacterial cell proteins is a valuable ~ilternative and has been successfully applied in rhizosphere studies of maize [23]. In the present study, we examined the microflora of witloof chicory leaves * Present address: IRRI Plant PathologyDepartment, P.O. Box 933, 1099 Manila, Philippines. ** Present address: Smith Kline-RIT,Rue de l'Institut, B-1330 Rixensart, Belgium.

176

M.F. Van Outryve et al.

(Cichorium intybus L. v a r . foliosum H e g i ) . T h e s e a r e a l o w c a l o r i e v e g e t a b l e , cultivated mainly in Belgium, France, and the Netherlands. The leaves are a r r a n g e d in h e a d s w h i c h a r e o b t a i n e d i n a t w o - s t a g e d p r o c e s s . T h e r o o t s a r e h a r v e s t e d 6 m o n t h s a f t e r s o w i n g in t h e field (stage 1) a n d m a y b e s t o r e d f o r s e v e r a l m o n t h s a t I~ o r f o r c e d i m m e d i a t e l y . F o r c i n g (stage 2) is p e r f o r m e d in the dark under conditions of standardized temperature and humidity, and results in etiolated heads known as witloof chicory. I t is n o t c l e a r w h e t h e r t h e l e a f m i c r o e n v i r o n m e n t o f t h e e t i o l a t e d c h i c o r y h e a d is t o b e c o n s i d e r e d as p h y l l o s p h e r e [24, 30], p h y l l o p l a n e [25], o r g e m m i s p h e r e [26]. I n fact, t h e c h i c o r y l e a v e s a r e a r r a n g e d i n a b u d , a m i c r o h a b i t a t t h a t h a s b e e n t e r m e d " g e m m i s p h e r e " [27]. W e e x a m i n e d t h e m i c r o f l o r a o f s e p a r a t e l e a v e s m e a s u r i n g u p t o 15 c m i n l e n g t h a n d 5 c m i n w i d t h . T h e o b j e c t i v e o f t h i s s t u d y w a s t o i n v e n t o r y t h e b a c t e r i a l flora a s s o c i a t e d w i t h c h i c o r y h e a d l e a v e s b y m e a n s o f S D S - P A G E o f t o t a l b a c t e r i a l cell p r o t e i n s . The isolates were tested for antifungal activity and for phytopathogenicity. The results were compared with two previous studies on the bacterial microflora o f c h i c o r y s e e d s [35] a n d r o o t s [37]. Materials and Methods

Samples The following chicory varieties were examined: Alba (sample 1), Zoom (sample 2), 1822 (sample 3), and Kulma (samples 4-8). Sample 1 was forced under 8 cm of soil enriched with manure and without additional heating. All other chicory heads were grown in hydroponic systems at 16"C and 95% relative humidity. The heads were ready for sale, i.e., about 3 weeks old. From each head, consecutive leaf pairs were sampled and examined.

Isolation of Bacterial Microflora Each pair of leaves was shaken in quarter strength Ringer solution (Oxoid) with 0.025% Tween 20 (Sigma) in an Incubator Shaker G25 (New Brunswick Scientific Co.) at 250 rpm for 30 min. In the case of the variety Alba, leaves 2 and 3 were transferred to a fresh washing solution after 30 min and shaken again for another 30 min. Only the second washing solution was used. Dilution series of these washing solutions were plated on the following media: nutrient agar (NA) (Difco) + 0.01% cycloheximide (Sigma; w/v) and 1/10 tryptic soy agar (TSA) (BBL) + 0.01% cycloheximide (w/v).. The number of bacteria/cm2 leaf surface, counted after 3 days incubation at 28~ was obtained by calculating the number of bacteria in the original washing Solution and dividing this number by twice (upper/lower side) the total leaf surface. Colonies were picked and purified from leaf pairs 2-3, 7-8, and 15-16 (sample 1); 15-16 (samples 2--4); and 7-8, 11-12, and 15-16 (samples 5-8). They were obtained in two ways: (1) in the case of samples 1-5, all colonies were picked from random plates (total screening); (2) in the other cases, only dominant colony types (colonies that occurred more than once on a plate) were selected (selective screening). Further purification was performed on NA. For prolonged conservation, overnight nutrient broth cultures (70%) were mixed with glycerol (30%) and conserved at -70~

Characterization of Isolates The isolates were grouped by visual comparison of their protein fingerprint types [23], defined as a set of visibly identical protein profiles obtained under standardized conditions. At least one isolate of each fingerprint type was further characterized by Gram's method, colony characteristics on

Bacteria on Chicory Leaves

177

Table 1. Quantitative data on chicory varieties, fingerprint types, antifungal activity, and phytopathogenicity of the chicory head leaf isolates Sample no./ variety

1/ Alba

2/ Zoom

3/ 4/ 5/ 6/ 7/ 8/ 1822 Kulma Kulma Kulma Kulma Kulma

No. of isolates No. of fingerprint types No. of isolates tested for antifungal activity No. of antifungal isolates No. of isolates tested for phytopathogenicity No. of phytopathogenic isolates

179 78

18 6

67 13

6 6

222 42

39 14

34 10

25 4

68 4

4 0

9 0

4 0

49 2

13 0

9 0

5 0

72 4

3 0

9 0

4 0

48 0

1i 0

8 0

6 l

NA, oxidase [29], reaction on Kligler Iron Agar [29] and/or OF-test [17], and fluorescence on King's medium B [21]. The gram-negative fermentatives and the oxidative, non fluorescent isolates were further identified by API 20E system or API 20NE system (API Systems, La Balme-lesGrottes, Montalieu Vercieu, France), respectively. We did not apply the latter system on fluorescent Pseudomonas. These were further differentiated by arginine dihydrolase [29] and gelatinase (Oxoid charcoal disks). The gram-positive isolates were characterized by cell morphology, motility, anaerobic growth on NA, spore stain [31], and catalase and oxidase reactions [29].

In vitro Antimicrobial Activity o f Isolates At least one strain from each fingerprint type was tested for the production of antifungal substances against Colletotrichum lindemutianum PGSF-58, Rhizoctonia solani PGSF-18, Botrytis sp. PGSF29, Sclerotinia sclerotiorum PGSF-55, Sclerotinia minor PGSF-87, and Phoma exigua PGSF-94. With exception of the first, all these fungi are chicory pathogens; the latter two were isolated from infected chicory roots. The antifungal activity was tested by incubating the test strains (four per plate) on potato dextrose agar (Difco) inoculated with a spore suspension of Colletotrichum lindemuthianum or, in the other cases, by inoculating the target fungus as a mycelium plug in the middle of a PDA-plate [4]. Inhibition zones around the streaked bacterial isolates were recorded after 3 days and reevaluated after 7 days incubation at 28~

Phytopathogenicity The phytopathogenicity of the isolates (at least one for each fingerprint type) was tested on etiolated head leaves (variety "Zoom") obtained from heads ready for sale. The leaves were wounded with an inoculated needle and incubated on sterile moistened filter paper in Petri dishes at 28~ The results, i.e., a well-delineated red rot around the inoculation site in case of positive infection, were observed after 24 hours and compared with known phytopathogenie isolates [36].

Results

Quantitative Analysis On variety Alba, forced with soil cover, the numbers of bacteria (+ standard e r r o r o f t h e m e a n ) p e r c m 2 l e a f s u r f a c e v a r i e d f r o m ( 2 . 0 + 0 . 5 ) x 10 3 o n l e a v e s 7 - 8 a n d (1.5 + 2 . 3 ) x 10 2 o n l e a v e s 1 1 - 1 2 t o (6.2 + 5.5) x 10 t o n l e a v e s 1 5 16. T h e d e c r e a s e b e t w e e n l e a f p a i r s 2 - 3 a n d 7 - 8 w a s s i g n i f i c a n t (t t e s t P =

178

M.F. Van Outryve et al.

Table 2. The number of fingerprint types and isolates (in brackets) of chicory leaf isolates

Isolates Gram-negative

Fermentative

Oxidative

Phenotype

API codea

Erwinia herbicolaEntero6acter agglomerans Erwinia sp. Serratia liquefaciens Fluorescent Pseudomonas Other Pseudomonas sp. Pseudomonas cichorii Pseudomonas cepacia Xanthomonas maltophtTia Flavobacterium sp. Achromobacter sp. Citrobacter sp. Alcaligenes denitrificans Oxidase positive Oxidase negative

1045573,1005573 b 1005133 b 5307763 b

0457777,0477345 c 0472345 c 0453204 c 1104513 c 1047777 ~

Gram-positive rods Gram-positive cocci Coryneforms To obtain the seven-digit code, the API 20E and API 20NE tests are grouped by three. One, two, and four marks are given in case of positive results in the first, second, and fourth test respectively; otherwise no marks are given. Each three marks are then added to give one of the digits of the code b The API 20E System consists of the following tests:/3-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, growth on citrate, H2S-formation, activity ofurease, tryptophane deaminase, indol formation, Voges Proskauer reaction, gelatinase, fermentation of glucose, mannitol, inositol, sorbitol, rhamnose, saccharose, melibiose, arnygdaline, arabinose c The API 20NE System consists of the following tests: NO3-reduction, formation ofindol, glucose fermentation, presence of arginine dihydrolase, urease, B-glucosidase, gelatinase, B-galactosidase, growth on glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate

0.05). N o b a c t e r i a w e r e o b t a i n e d f r o m l e a f p a i r s 1 9 - 2 0 a n d 23 t o t h e m i d d l e of the head. On the heads obtained from hydroponic systems, the decrease was m o r e g r a d u a l . O n l e a v e s 7 - 8 , t h e n u m b e r s f l u c t u a t e d b e t w e e n (2.9 + 0.2) x 105 ( s a m p l e n u m b e r 6) a n d (1.6 + 0.1) x 104 ( s a m p l e n u m b e r 5). O n l e a v e s 1 1 - 1 2 , t h e n u m b e r s r a n g e d f r o m (6.8 _+ 0.7) x 105 ( s a m p l e n u m b e r 7) t o (3.2 _+ 0.2) x 103 ( s a m p l e n u m b e r 4). F o r l e a v e s 1 5 - 1 6 , t h e b a c t e r i a l c o u n t s r a n g e d f r o m (8.4 _+ 0.6) • 104 ( s a m p l e n u m b e r 6) t o (4.4 _+ 4.2) x 10 t ( s a m p l e n u m b e r 4). I n all s a m p l e s e x c e p t s a m p l e 2, a s i g n i f i c a n t d e c r e a s e w a s f o u n d b e t w e e n l e a f p a i r s 7 - 8 , 1 1 - 1 2 , a n d 1 5 - 1 6 . I n s a m p l e 2 (i.e., v a r i e t y Z o o m ) , t h e d e c r e a s e was significant between leaf pairs 11-12 and 15-16, but not between 7-8 and 11-12.

Characterization o f the Fingerprint Types I n t o t a l , 590 i s o l a t e s w e r e o b t a i n e d a n d g r o u p e d i n t o 149 f i n g e r p r i n t t y p e s designated CH. The number of isolates per head and the number of fingerprint

179

Bacteria on Chicory Leaves

T a b l e 2.

I/ Alba --

Extended

2/ Zoom

3/ 1822

1 (6)

1 (11)

1 (6)

Sample no./variety 4/ 5/ Kulma Kulma 1 (1)

1 (5)

.

.

9-(85)

1 (2) --

---

1 (1) --

---

-1 (3)

---

1 (14)

.

.

.

26 (26) 9 (9) 24 (26) 3 (3) 3 (3)

.

1 (2)

.

.

.

.

1

.

1 1

1 (13) 1 (10)

---

---

---

2 1

1 (3)

2 (6)

1 (1)

--

3

l

--

--

--

1

1 (1)

--

2

2 (2)

.

.

(7)

.

.

l

12 (20)

3 (3)

1 (1)

1 (1)

3 (4)

--

4 (4)

3 (3)

2 (2)

--

--

4 (4) 1 (1)

1 (1) -

4 (4)

1 (1) l (l)

1 (1) -

--

1 (1)

--

1 (1)

1 (4)

--

1 (1)

--

-

20 1

1

. .

2 (2)

.

2 (25) 1 (2)

.

2 (39)

Total no. 8/ of fingerKulma print t y p e s

3 (18) 1 (7)

2 (6) 1 (21)

.

7/ Kulma

.

15 ( 1 3 8 ) 1 (15)

--

.

. .

.

--

--

.

.

.

.

--

--

1 (3) 1 (2)

.

.

--

.

.

1 (5)

.

.

6/ Kulma

38 19 35 5

7

types distinguished a m o n g the isolates f r o m each h e a d are presented in T a b l e I. T a b l e 2 shows the occurrence o f the different p h e n o t y p i c groups. O n l y the fingerprint type C H 0 0 1 (175 isolates), identified as a fluorescent Pseudomonas, was repeatedly r e c o v e r e d f r o m several heads. It was f o u n d on all s a m p l e s except on the single h e a d o f variety 1822. It constituted between 30 a n d 50% o f the isolates o f s a m p l e s 1, a n d 5-8, a n d was o b t a i n e d in smaller n u m b e r s (11 a n d 16%) f r o m leaf pairs 15-16 o f s a m p l e s 2 a n d 4, respectively. T h e fingerprint types C H 0 2 3 (23 isolates) a n d C H 2 3 7 (34 isolates) were f o u n d on all heads o f the variety K u l m a forced on a h y d r o p o n i c s y s t e m except on K u l m a s a m p l e 4. T h e f o r m e r was identified as a fluorescent Pseudomonas, the latter fingerprint was identified as "Pseudomonas paucimobilis" b y the A P I 2 0 N E system. This last identification is questionable as the isolates were p i n k on N A a n d no p i n k Pseudomonas paucimobilis are known. T w e l v e o t h e r fingerprint types were f o u n d on at least two chicory heads. A m o n g these we identified fluorescent Pseudomonas, Erwinia herbicola-Enterobacter agglomerans, Erwinia sp., a n d Flavobacterium sp. T h e m a j o r i t y o f the total n u m b e r o f fingerprint types (90%), however, was f o u n d only once.

Pigmentation of Isolates T h e m a j o r i t y o f the isolates (74%) o b t a i n e d f r o m all heads was a c h r o m o g e n i c . T h i r t e e n percent o f the isolates were yellow. T h e y were identified as Pseudomonas cepacia, Citrobacter sp., Xanthomonas maltophilia, a n d Erwinia sp.

180

M.F. Van Outryveet al.

Nine percent were pink. A few isolates (P. cepacia or Flavobacterium sp.) were orange, and one was violet.

Comparison of Isolation Methods Two isolation procedures were used in this study.

Total Screening Procedure (samples 1-5). All colonies were picked randomly from plates. These 492 isolates were grouped into 134 fingerprint types, of which 108 (126 isolates) occurred only on one though not the same leaf pair. This means that 74% of these isolates can be qualified as "occasional colonizers." Selective Screening Procedure (other samples). Only visually dominant colony types were isolated. This procedure considerably reduced the amount of work without losing the important information on frequently occurring protein profiles. The 99 isolates were grouped into 27 fingerprint types of which only 11 (11 isolates) were obtained from only one though not the same leaf pair. These "occasional" isolates represent 11% of the total number of isolates obtained during the selective screening. The quantitatively most important fingerprint types CH001, CH023, and CH237 were recovered in large numbers by both screening procedures. Two isolation media were used: NA and 1/10 TSA, both supplemented with 0.01% cycloheximide. Twenty of the 29 fingerprint types that occurred on more than one leaf pair were isolated from both media. Nine fingerprint types were obtained from one medium only. Among the latter we found the two Flavobacterium fingerprint types isolated from 1/10 TSA only and the two fingerprint types identified as Citrobaeter sp. and Serratia liquefaciens isolated from NA only. All isolates, however, were able to grow on both isolation media.

Antifungal lsolates One hundred sixty-one isolates (at least one for each fingerprint type) were tested for antifungal activity against six fungal phytopathogens. Only 3% of the fingerprint types contained antifungal isolates. The six antifungal isolates thus obtained belonged to five different fingerprint types. They were identified as fluorescent Pseudomonas (four isolates, belonging to three different fingerprint types and with distinct spectra ofantifungal activity), Serratia liquefaciens (one isolate), and one gram-positive asporogeneous isolate. The activities of the isolates that inhibited more than one fungus are given in Table 3.

Latent Phytopathogenic Isolates Five isolates of the 161 tested (at least one for each fingerprint type) were able to produce symptoms upon inoculation of chicory leaves. They all were identified as fluorescent Pseudomonas and belonged to five different fingerprint

Bacteria on Chicory Leaves

181

Table 3, Isolates of the chicory seed microflora ~ rhizosphere ~, and phylloplanec: antagonistic activity against fungi

Isolate no.

Fingerprint type

PGSB-7366 o PGSB-7452 a PGSB-7484 ~ PGSB-7490 ~ PGSB-7508 a PGSB-7213 b PGSB-7218 b PGSB-7223 b PGSB-7293 b PGSB-7299 b PGSB-7333 ~ PGSB-7457 b PGSB-7459 b PGSB-7470 b PGSB-7562 b PGSB-7567 b PGSB-6593 c PGSB-6620 c PGSB-6647 c PGSB-6888 c PGSB-6912 c

CH382 CH407 CH407 CH386 CH386 CH054 CH002 CH057 CH003 CH009 CH001 CH034 CH212 CH201 CH002 CH001 CH347 CH001 CH377 CH287 CH018

Fungus a Identification

F29 F18 F55 F58 F87 F94

E. herbicola

+

+

+

+

+

+

sp. B a c i l l u s sp. Gram-negative Gram-negative Gram-positive Fluorescent P s . B a c i l l u s sp. Fluorescent P s . Fluorescent P s . Fluorescent Ps. Gram-negative Fluorescent P s . Gram-positive Fluorescent P s . Fluorescent P s . Gram-positive Fluorescent P s .

+ + + + +

+ _ + + -

+ + + + +

+ + + + +

+ + + + +

+ + + + -

Bacillus

+

.

+

+

.

+

.

-

.

.

+

-

+

+

-

+

+

+

+

-

-

+

+

-

-

+

.

.

-

+

.

.

+

+

+

-

+

-

+

+

+

-

+

-

-

+

+

-

-

-

+

+

+

-

.

.

.

.

+

+

+

-

+

+

+

+

+

+

+

+

S. liquefaciens

+

+

+

+

+

-

Fluorescent Fluorescent

Ps.

+

+

-

+

+

-

Ps.

+

-

-

+

+

-

d F29:

B o t r y t i s sp., FIB: R h i z o c t o n i a s o l a n i , F55: S c l e r o t i n i a s c l e r o t i o r u m , F58: Colletotrichum lindemuthianum, F 8 7 : S c l e r o t i n i a m i n o r , F94: P h o m a e x i g u a Ps. = P s e u d o m o n a s

types of this species. Four of them were isolated from Alba forced under c o v e r ( s a m p l e 1).

soil

Discussion I n t h i s s t u d y , a b o u t 105 b a c t e r i a p e r c m 2 w e r e f o u n d o n t h e o u t e r c h i c o r y h e a d leaves. The gradient of microorganisms towards the inner leaves of the chicory heads differed between the two forcing methods: the decrease in numbers of bacteria per cm 2 leaf surface was more gradual on the heads obtained on hydroponic systems than on the head obtained with soil cover. The heads forced with soil cover are more closed due to the soil pressure on them. The heads cultivated on hydroponic systems are not so compact and bacteria can more easily colonize the inner leaves. I n o u r a n a l y s i s , g r a m - n e g a t i v e i s o l a t e s ( 1 0 5 i s o l a t e s , 101 f i n g e r p r i n t t y p e s ) were predominant. However, gram-positive bacteria comprised 17% of the isolates obtained from Alba, forced with soil cover, Gram-positive bacteria constitute an important part of the soil microflora which can contain up to 60% arthrobacter and Bacillus sp. [2]. F r o m t h e c h i c o r y h e a d s o b t a i n e d o n hydroponic systems, less gram-positives were isolated (0-8%).

182

M.F. Van Outryveet al.

A few bacterial species are dominant on the leaf surface [3, 7, 14]. In our study, only 10% of the fingerprint types contained more than one isolate. The fingerprint type CH001, identified as a fluorescent Pseudomonas, was found on all but one head. Fluorescent pseudomonads in general were predominant on the heads obtained by both forcing methods. Among the 278 fluorescent Pseudomonas isolates, 20 fingerprint types could be distinguished. Heterogeneity within the fluorescent pseudomonads has also been demonstrated for isolates in the rhizosphere of maize where 27 distinct protein fingerprint types were identified [23]. The fluorescent Pseudornonas isolates from the chicory leaves can partly originate from the roots, as the fingerprint types CH001 and CH002, isolated in high numbers from the heads, also predominated in the rhizosphere of chicory plants [37]. The roots were stored for weeks, enabling contact with the dormant buds. Fluorescent Pseudomonas isolates may also originate from organic substances in the soil, which are frequently colonized by fluorescent pseudomonads [5]. Yellow pigmented residents of plants have often been lumped together by microbial ecologists [see 9], although they constitute a taxonomically and physiologically heterogeneous group [15]. Xanthomonas maltophilia, one of the yellow pigmented species found in the present study, was identified as a phyllosphere inhabitant of the chicory head under soil cover. The same species was frequently isolated from the chicory rhizosphere [37]. There is no proof that the Xanthomonas maltophilia phylloplane isolates originate from the rhizosphere as both ecosystems harbor Xanthomonas maltophilia strains with different fingerprint types. Three other species were restricted to the head under soil cover: Citrobacter sp., Alcaligenes denitrificans, and Serratia liquefaciens, the former two being well-known soil bacteria. The soil is clearly a source of many of the phylloplane isolates. The 18 Erwinia herbicola-Enterobacter agglomerans isolates (1 fingerprint type) from three different chicory heads obtained on hydroponics were all cream-colored. Erwinia herbicola has been reported as a typical leaf inhabitant [33], and the phenotypic features of the species were extensively studied by Verdonck et al. [39]. A dominant group of chromogenic and achromogenic Erwinia herbicola-Enterobacter agglomerans isolates (but with different fingerprint type) was obtained from chicory seeds [351. Crosse [ 10] demonstrated that Pseudomonas syringae pv. morsprunorum can be isolated from healthy leaf tissue. This phenomenon has since been reported for several plant pathogenic bacteria. Five witloof chicory isolates were able to develop disease symptoms upon inoculation; four of these were obtained from the head forced under soil cover. This suggests that the soil contains an important stock of potential phytopathogenic bacteria which only affect wounded plants [36]. Thus, it is important to handle the heads carefully to limit injury. Few leaf isolates (4%) were clearly active against phytopathogenic fungi tested. One antifungal isolate was identified as Serratia liquefaciens, a well-known insect pathogen. Its pathogenicity is correlated with the production ofproteinase and chitinase [ 16]. Chitinase can also affect fungi which contain chitin in their cell walls. None of the 24 Pseudomonas cepacia strains isolated in this study showed any antifungal properties. This species is known as an inhibitor of fungi [19, 25]. Fluorescent pseudomonads are well-known producers of secondary metabolites with antifungal activity [28].

Bacteria on Chicory Leaves

183

Table 4. The frequently occurring fingerprint types found on chicory seeds, roots, and head leaves

Identity Fluorescent Pseudomonas Fluorescent Pseudomonas Alcaligenes paradoxus Pseudomonas sp. Erwinia herbicolaEnterobacter agglomerans Arthrobacter

Fingerprint type

Occurrence on chicory~ Seedsb Roots c Leaves (%) (%) (%) 9 13 12

CH001 CH002 CH213 CH237 CH382 CH383

30 6

36 16

Percentage of isolates obtained from the specified environment b Van Outryve et al. [35] c Van Outryve et al. [37]

Microbial ecological studies, based on indirect observations, suffer from a few shortcomings inherent to their methodology. T h e isolation technique, as well as incubation conditions, greatly influence the organisms obtained [1 1]. Direct isolation m e t h o d s are r e c o m m e n d e d in cases where precision is essential [11, 13]. T h e i r shortcomings are their tediousness and the fact that the identification is in m o s t cases impossible [13]. Hence, studies in microbial ecology always represent a c o m p r o m i s e between the objectives and the tools necessary to achieve t h e m [ 11 ]. In the present study, general media and incubation conditions were used throughout. Thus, specialized groups o f microorganisms might have been missed, e.g., streptomycetes which were only occasionally obtained. In a first stage o f the study on the bacterial flora o f chicory, the isolates were characterized by their protein profiles obtained by S D S - P A G E o f whole-cell proteins. T h e expression o f the microbial g e n o m e results in the synthesis o f about 2,000 different protein molecules that compose the microbial cell [18]. Bacteria with similar protein profiles are closely related [20]. O n f o r m e r occasions, we have m e n t i o n e d that several reproducible fingerprint types could be distinguished within one bacterial species, not only for fluorescent Pseudomonas but also for Erwinia herbicola-Enterobacter agglomerans, Agrobacterium radiobacter, Pseudomonas paucimobilis, and for Xanthomonas maltophilia [35, 37]. I m p o r t a n t characteristics like phytopathogenicity could generally not be correlated with a particular fingerprint type. W h e n tested upon artificial inoculation, 22% o f the fluorescent Pseudomonas fingerprint types c o n t a i n e d pathogenic as well as nonpathogenic isolates. This p h e n o m e n o n has been reported for other phytopathogenic bacteria [34, 38]. Similar findings were obtained concerning the antifungal activity including isolates with antagonistic activity against m o r e than one fungus (Table 3). Usually very few isolates showed strong antifungal activity, while other isolates with the same fingerprint type sometimes exhibited activity against one fungus or even none. F o r further identification o f the isolates, we extensively used API Systems which have been developed especially for clinical purposes. Careful interpre-

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tation of the API profiles is necessary, however, as misidentifications may occur [32]. An important shortcoming of the API System is that the majority of the API profiles remain unidentified, even after consultation of the API computer service. In two previous publications we described the microflora of chicory seed [35] and roots [37]. Both microfloras contained a majority of gram-negative isolates. This was also the case for the chicory phylloplane microflora. Within these populations, low numbers o f antifungal or phytopathogenic isolates occurred. Each of the three ecosystems was characterized by frequently occurring fingerprint types (Table 4). These organisms can be considered as "resident" [26], i.e., they live and multiply in that ecosystem. The majority of the fingerprint types, on the other hand, represent the accidental microflora or casuals [26]. By means of the frequently occurring fingerprint types, we were able to define a general picture of the seed microflora, rhizosphere, and phylloplane of chicory. However, it was not possible to distinguish details between, e.g., different varieties, roots from one sampling, leaves from one head, or even different heads. The seed microflora, which was obtained from dry-stored seeds, was characterized by frequent occurrence of Erwinia herbicola-Enterobacter agglomerans and arthrobacter. The approximate minimal density of these major seed fingerprint types ranged up to 103 CFU/g seed. We found no obvious reason why Erwinia herbicola was present in such high numbers on stored chicory seed. This species was also abundantly present on weed seeds collected from the field [22]. There are indications that arthrobacter can survive in environments poor in nutrients [6, 8]. By careful examination of the occurrence of the fingerprint types, we were able to show the inhibiting influence of seed germination on arthrobacter and not on the erwinias. This phenomenon has to be considered when seed bacterization is used as a biological control treatment, as it can play a part in the establishment o f the early rhizosphere microflora. We were not able to show a relationship between the seed microflora and the rhizosphere of the seedlings in the field. The chicory rhizosphere was characterized by two different fluorescent Pseudomonas fingerprint types (CH001 and CH002) and a fingerprint type identified as Alcaligenes paradoxus; the latter species occurred mainly on younger roots. Until now, it was isolated from soil only by specialized enrichment conditions [1, 12]. The Alcaligenes paradoxus isolates were inhibited in vitro by isolates of fluorescent Pseudomonas fingerprint type CH001 which were obtained from older roots. As indicated above, the two fluorescent Pseudomonas fingerprint types (CH001 and CH002) were also found on chicory heads. The approximate minimal densities of the fluorescent Pseudomonas fingerprint types CH001 and CH002 ranged from 102 to 104 C F U / g root and from 101 to 10 2 C F U / c m 3 leaf surface. We have already pointed out that the correlation between root and leaf microflora composition can partially be explained by their intimate contact during root storage. PAGE allowed a quick and efficient characterization of the microflora of chicory, i.e., the screening of a large number of isolates (184 seed isolates, 233 root isolates, 590 leaf isolates). The microflora associated with the different plant parts as well as environmental influences on that microflora were deter-

Bacteria on Chicory Leaves

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m i n e d ; h o w e v e r , it w a s n o t p o s s i b l e t o o b t a i n m o r e d e t a i l e d i n f o r m a t i o n . T h i s c h a r a c t e r i z a t i o n t e c h n i q u e is a first s t e p t o a n u r g e n t l y n e e d e d , m o r e c o m p r e hensive a p p r o a c h in bacterial ecology.

Acknowledgments. We thank Ir. Sarrazijn (Provinciaal Onderzoek- en Voorlichtingscentrum voor Land- en Tuinbouw, Beitem - Rumbeke) for providing the plant material. M. F. Van Outryve is indebted to the Instituut tot Aanmoediglng van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL Belgium) for a scholarship. J. Swings is indebted to the Nationaal Fonds voor Wetenschappelijk Onderzoek (NFWO Belgium) for research grants.

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The bacterial microflora of witloof chicory (Cichorium intybus L. var.foliosum Hegi) leaves.

The bacterial flora on the heads of four different witloof chicory varieties was examined. The 590 isolates were characterized by their SDS-PAGE prote...
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