Infection Sites of Pseudomonas Cichorii Into Head Leaf of Lettuce

日植病報 62: 125-129 (1996)

Ann. Phytopathol. Soc. Jpn. 62: 125-129 (1996)

Infection Sites of Pseudomonas cichorii into Head Leaf of Lettuce

Yasufumi HIKICHI*,Akira SAITO*and Kazumi SUZUKI

Abstract Sectionsof middlehead leaf of lettucewere stained with fluoresceinisothiocyanate-conjug antibodyagainst Pseudomonas cichorii, a causalagent of bacterialrot of lettuce,and wereobserve undera fluorescentmicroscope. Fluorescent antibody-labeled P. cichorii cells were observed in stomat of the sections,which did not contain browing symptom of the disease.Special fluorescent specks deriv fromP. cichoriiwere most frequently observed in guardcells and intercellularspaces of substoma cavities.Browing, which was the initialsymptom of disease,progressed from epidermis to mesophl In advanceof progressof browingsymptom, specific fluorescent specks were observedin intercellu spacesof epidermisand then were observed in intercellularspaces of mesophyll.These results sugge that P. cichoriifirst invaded head leaf of lettucethrough stomata, later multiplied in intercellularspace of epidermisand colonizedintercellular spaces of mesophyll.Specific fluorescent specks were also observedin leafhairs. (ReceivedSeptember 1, 1995;Accepted December 27, 1995) Keywords: Pseudomonascichorii, lettuce, head leaf, stomata infection, intercellular space.

INTRODUCTION MATERIALS AND METHODS

Bacterial rot of lettuce caused by Pseudomonas ci- Plants and bacteria. Seedlings of lettuce chorii results in large losses and is prevalent in spring- (Lactuca sativa L. cultivar Success) were transplanted cropping type of lettuce in north Japan, the summer- into pots (755•~423•~170mm) 21 days after sowing, and cropping type in the highlands and the autumn-cropping were cultivated in a greenhouse. Lettuces in the middle type in the lowlands14,20). The disease, referred to by stage of head formation were inoculated with P. cichorii growers as varnish spot in California and tar in Japan, SPC9001, which was obtained from Mr. T. Shirakawa, is characterized by shiny, dark-brown, firm and necrotic Morioka Branch, National Research Institute of Vegeta- spots that occur on leaves underneath the second or bles, Ornamental plants and Tea, at the bacterial den- third outermost head leaves (Fig. 1)5,14,20)The primary sity of 106 colony forming unit (cfu)/ml by a spray infection sources seem to be seeds, infected plant debris, application (50ml/m2). This bacterial strain was iso- weeds and soil, and the bacteria have survived in soil lated from lettuce plants cultivated in Ichinohe, Iwate contaminated with infected lettuce debris for more than Prefecture, Japan and was shaking-cultured in YP- three months4,11,15,21).Shirata et al.19) have reported that medium (bacto-yeast extract, 5g; bacto-peptone, 10g; lesions in unwounded leaf of lettuce inoculated with P. deionized water, 1 liter; pH 6.8) for 24hr at 30•Ž. Soil cichorii elongate from stomata and hairs. However, and the infected lettuces were mixed in the pots at infection sites of P. cichorii into head leaf of lettuce are harvest time and were kept for one month in the green- still poorly understood. house. Then seedlings of lettuce 21 days after sowing Detection of plant-pathogenic bacteria in host plants were transplanted into the pots containing the soil and with immunofluorescent antibody is useful for studies on the infected lettuce debris and were cultivated in the infection route of bacteria into host plants and popula- greenhouse until maturity. Pots were watered one time tion of bacteria in the host plants8,9,22). a day by a spray application (300ml/m2). In the present paper we investigated the invasion sites Preparation of IgG8,9,). P. cichorii SPC9001 was of head leaf of lettuce by P. cichorii and the bacterial shaking-cultured in YP-medium for 24hr at 30•Ž, multiplication sites in head leaf by using fluorescein suspended into 0.01M phosphate buffer containing isothiocyanate (FITC)-conjugated antibody against P. 0.85% NaCl (PBS, pH 7.0), washed twice by centrifuga- cichorii. tion (5000rpm, 10min), and the final precipitate was resuspended into 4ml of PBS with 50% FCA at a con-

centration of ca. 108 cells/ml. Antiserum against P.

* Iwate Biotechnology Research Center , Narita 22-174-4, Kitakami 024, Japan 岩手生物工学研究センター 126 日本植物病理学会報第62巻第2号平成8年4月

cichorii SPC9001 was produced in a rabbit by adminis- tering four intramuscular injections at the bacterial density of ca. 108 cells/ml/rabbit over two months. The rabbit was bled 10 days after the final injection. Anti- body titer against P. cichorii was 1/5120 in the micro- precipitin test. Ten ml of antiserum was added to 40ml of PBS and 50ml of water-saturated ammonium sulfate. That was incubated at 0•Ž for 60min and was centrifuged at 8000rpm for 15min. The precipitate was resolved in 100ml of PBS and was added to 50ml of Fig. 1. Bacterial rot of lettuce cultivated in the pot of water-saturated ammonium sulfate. That was incubated green house, caused by Pseudomonas cichorii. at 0•Ž for 10min and was centrifuged at 8000rpm for 15 min. The precipitate resolved in 10ml of PBS was leaves underneath the second or third outermost head dialyzed overnight, and IgG was obtained. leaves (Fig. 1). Preparation of FITC-conjugated antibody8,9). Middle head-leaves, which had no symptom, were cut

One ml of IgG was added to 100ml of 0.02M Na2CO3- into cross sections. In observation under a light micro- NaHCO, buffer (pH 9.8) containing 0.011% FITC and scope, any browning symptom of the disease was not was incubated at 0•Ž for 6hr. By gel permeation with observed. Specific fluorescent specks derived from P. Sephadex G-25 (Pharmacia) FITC-conjugated antibody cichorii were prevalently observed in stomata of sec- was obtained. tions under a fluorescent microscope (Fig. 2). They were Removal of nonspecific antiboy8,9). Lettuce specially observed in guard cells and intercellular spaces plants at the weight of 200g were ground in acetone of substomatal cavities. at -20•Ž with a mortar and pestle. That was washed in Multiplication sites of P. cichorii in head leaf acetone and was suspended in 80% ethanol solution. of lettuce Then that was incubated at 80•Ž for 10min and was Middle head-leaves, which had rot symptom, were cut centrifuged at 8000rpm for 10min. The precipitate was into cross sections. Browing, which was the initial symp- suspended in PBS and was centrifuged at 8000rpm for tom of disease, was first observed in epidermis and later 10min. The same manipulation was done three times. progressed into the mesophyll under the light micro- The precipitate was added PBS containing FITC- scope (Fig. 3-A, E, G and I). In observation under a conjugated antibody and 0.005% NaN3 and was incubat- fluorescent microscope, specific fluorescent specks de- ed at room temperature overnight. Then that was rived from P. cichorii were observed in intercellular centrifuged at 3000rpm for 5min. The supernatant was spaces of epidermis (Fig. 3-B, D and F). Then they were the FITC-conjugated antibody against P. cichorii which observed in intercellular spaces of mesophyll (Fig. 3-D, was removed nonspecific antibody binding to antigen F, H and J). Specific fluorescent specks were successive- derived from healthy lettuce plants. ly observed in stoma and intercellular spaces of epider- No specific fluorescent speck was observed when this mis and mesophyll (Fig. 3-C and D). When specific FITC-conjugated antibody was incubated with P. mar- fluorescent specks were observed in intercellular spaces ginalis pv. marginalis YM8113 and P. viridiflava of epidermis and mesophyll, browing developed in the YM7926, which were obtained from Mr. T. Shirakawa, mesophyll. P. glumae PG-47) and P. plantarii1) MAFF301723. Specific fluorescent specks derived from P. cichorii Fluorescent microscopic observation8,9). Middle were also observed in leaf hairs (Fig. 4). head leaves of lettuce at harvest were cut into small pieces (10•~20mm) and fixed in 0.5% (v/v) glutaralde- DISCUSSION hyde in PBS for 2-3hr. They were washed in PBS three times and were used to obtain sections at the thickness Most plant-pathogenic bacteria do not possess inva- of 150ƒÊm with an Automatic MT-3 (Nippon Medical & sive abilities into host plants. They are known to pene- Chemical Instruments). The sections were dipped in trate their host plants through damaged surfaces or FITC-conjugated antibody against P. cichorii at 37•Ž for natural openings (stomata and hydathodes) in stems or 15min and washed in PBS three times. Sections were leaves2,3,6,11-13,16-19).Infection frequency of the epiphytic observed under a fluorescent microscope (Nikon EFD2, bacteria into host plants and disease incidence are excitation length: 490nm, emmision length: 525nm). related to the bacterial density on host plants, stomatal numbers and stomatal pore size 16). P. syringae pv.

RESULTS phaseoricola cells preferentially attach to stomata of bean leaves17). We have indicated that P. cichorii exists Stomatal infection of P. cichorii into head on lettuce leaves as the epiphytic bacterium, and bacte- leaves of lettuce rial rot is detected on lettuce leaves, from which P. Shiny, dark-brown, firm and necrotic spots, which cichorii is isolated at the density of above 105 cfu/g fresh were similar to those occurred in fields, occurred on weight10). In this study, P. cichorii cells were observed in Ann. Phytopathol. Soc. Jpn. 62 (2). April, 1996 127

Fig. 2. Stomatal infection of Pseudomonas cichorii into head leaf of lettuce. Cross sections of middle head leaf of

lettuce in the harvesting time were stained with fluorescein isothiocyanate-conjugated antibody against P.

cichorii and were observed under a light microscope (A, •~400; C, •~400; E, •~400 and G, •~100) and a

fluorescent microscope (B, •~400; D, •~400; F, •~400 and H, •~100). The arrows indicate the specific fluorescent

specks derived from P. cichorii. S, stoma; G, guard cell; SC, substomatal cavity. stomata of non-diseased head leaves of lettuce. They not show them experimentally. In this study, P. cichorii were observed in guard cells and intercellular spaces of invaded head leaves of lettuce through stomata, first substomatal cavities, indicating that P. cichorii invaded multiplied in intercellular spaces of epidermis, and later into head leaves through stomata. The previous and multiplied in intercellular spaces of mesophyll. Along present results suggested that population of P. cichorii with the bacterial multiplication, browning also prog- on head leaf was an important causal factor of in- ressed from epidermis to mesophyll. fectivity of P. cichorii into head leaf of lettuce. P. cichorii cells were observed in leaf hairs, but their Most plant-pathogenic bacteria, which invade host role in symptom development was not clear from the plants through stomata, multiply in intercellular spaces present study. of substomatal cavities and continuously multiply in intercellular spaces of parenchyma and mesophyll2,11,12,18). We wish to thank Mr. T. Shirakawa, Morioka Branch, Infected host cells are gradually damaged11). Shirata et National Research Institute of Vegetables, Ornamental al.19) have speculated on the invasion site and multiplica- plants and Tea, for his providing the bacterial strains. tion site of P. cichorii based on symptoms, but they do 128 日本植物病理学会報第62巻第2号平成8年4月

Fig. 3. Multiplication sites of Pseudomonas cichorii in head leaf of lettuce. Cross sections of middle head leaf of

lettuce in the harvesting time were stained with fluorescein isothiocyanate-conjugated antibody against P.

cichorii and were observed under a light microscope (A, •~100; C, •~400; E, •~100; G, •~100 and I, •~400) and

a fluorescent microscope (B, •~100; D, •~400; F, •~100; H, •~100 and J, •~400). The arrows indicate the specific

fluorescent specks derived from P. cichorii. E, epidermis; S, stoma; M, mesophyll; BR, browing which was the

initial symptom of bacterial rot of lettuce.

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