日 植 病 報61: 439-443 (1995) Ann. Phytopathol. Soc. Jpn. 61: 439-443 (1995)

Survival and Characteristics of Ice Nucleation-active Bacteria on Mulberry Trees ( spp.) and in Mulberry Pyralid ( pyloalis)*

Kyoichi TAKAHASHI**,***, Kenji WATANABE** and Mamoru SATO**

Abstract

Survival and population proportion of ice nucleation-active (INA) bacteria on mulberry tree and in an , mulberry pyralid, were investigated. Two groups of epiphytic bacteria, Pseudomonas syringae and Erwinia herbicola group bacteria formed dominant bacterial floras on mulberry leaves. E. herbicola group bacteria also distributed in most larvae of mulberry pyralid, but P. syringae was detectable in only some of them. Population frequency of INA P. syringae against total P. syringae in mulberry trees was very high (48.1%), whereas that of E. herbicola group bacteria was low (0.5%). Two group of INA bacteria were identified as P. syringae pv. mori and as Erwinia ananas which has not been detected from mulberry. These two species of INA bacteria were also first found in the insect, mulberry pyralid. A relationship between population of the INA bacteria and frost damage of mulberry trees was discussed. (Received January 25, 1995; Accepted May 18, 1995)

Key words: ice nucleation-active bacteria, Pseudomonas syringae pv. mori, Erwinia herbicola, Erwinia ananas, mulberry pyralid, mulberry trees.

syringae surviving on mulberry trees have not been INTRODUCTION detected and also there has been no information on the survival of INA bacteria in feeding mulberry Frost formation in plants requires the presence of leaves. suitable ice nucleus. Maki et al. found out that Pseudo- This paper deals with the results of the survival and monas syringae harbored an ability as ice nucleus11), and identification of INA bacteria on mulberry trees and in Arny et al. have shown that ice nucleation-active (INA) the insect, mulberry pyralid (Glyphodes pyloalis). bacteria are involved in frost damage to plants1). INA bacteria are widely distributed on plants in nature. MATERIALS AND METHODS Lindow et al. have found that many species of plant sampled from several locations harbored INA Sampling procedure of bacteria. Leaf samples bacteria10). Many species of INA bacteria such as P. were collected from mulberry trees (cv. Shin-ichinose, syringae pathovars, P. fluorescens, P. viridiflava, Xantho- cv. Kainezu and so on) at several locations of the field of monas campestris pv. translucens, Erwinia herbicola, E. National Institute of Sericultural and Entomological ananas and E. uredovora have been reported1,3,4,10,12). Science (NISES) located in Tsukuba-shi, Ibaraki, Japan. Moreover, distribution and population dynamics of INA Several leaves were collected from an upper position of bacteria have been investigated in many crops, fruit shoots, mixed each other and used as one sample. The trees and tea trees5,10,12). samples were washed vigorously in sterile distilled Survival of INA bacteria on mulberry trees (Morns water (20ml per one gram of fresh leaves) for 30min on spp.) have been described by Takahashi's research a rotary shaker. Leaf washing were plated on modified group13,16,18,19),and the INA bacterium was identified as LB medium (polypeptone 10g, yeast extract 5g, NaCl 10 P. syringae pv. mori which is the pathogen of bacterial g, agar 15g, distilled water 1 liter) and cultured at 25•Ž. blight of mulberry. Moreover, some properties such as From frost-damaged buds and leaves, the bacteria were plasmid profiles14) and the effect of temperature treat- isolated by homogenizing them with a mortar. Bacterial ment on ice nucleation activity in the bacteria8) were strains isolated were preserved by suspending cells in reported. However, INA bacterial species other than P. skim milk buffer (skim milk 10g, sodium L-glutamate

*This research was supported by a Grant -in-aid from Ministry of Agriculture , Forestry and Fisheries. ＊＊National Institute of Sericultural and Entomological Science , Tsukuba 305, Japan蚕 糸 ・昆 虫 農 業 技 術 研 究 所 ＊＊＊Present address: Kanagawa Prefectural Sericultural Experiment Station , Ebina 243-04, Japan現 在:神 奈 川 県 蚕 業 セ ン タ ー 440 日本植物病理学会報 第61巻 第5号 平成7年10月

1.5g, distilled water 100ml) and stored at -30•Ž until INA assay. RESULTS Larvae of Glyphodes pyloalis were collected from mulberry trees at several locations of NISES fields. Survival of INA bacteria on mulberry trees Whole body of the insect larvae was homogenized in Population of epiphytic bacteria surviving on mul- sterile distilled water using a homogenizer or a mortar berry leaves was surveyed at several days intervals after surface-sterilizing by 10% of sodium hypochlorite during October to November in 1993. Two (or three) solution (active chlorine, 8.5% to 13.5%). Mixtures were species of bacteria formed dominant flora on mulberry plated on modified LB medium and cultured at 25•Ž. leaves. These bacteria were identified as P. syringae pv. Assay of ice nucleation activity. Bacteria mori or E. herbicola group bacteria (E. ananas and E. grown on modified LB medium for 1 to 2 days at 20•Ž herbicola) as described later. E. herbicola group bacteria were used for INA assay. Ice nucleation activity was were detectable from all samples (52/52) and P. syringae determined by droplet freezing method on chromium was detected from 90per cent of the samples (47/52). plating copper plates described by Takahashi17). Ten They were observed at a high population on mulberry drops, each of 10ƒÊl, were placed on the plate at con- trees as shown in Fig. 1. Although several other kinds of trolled-temperature (-5•Ž). If all drops freezed within 60sec, the strain were judged to be INA bacteria. Identification of bacteria. Identification of Table 1. Proportion of ice nucleation-active (INA) Erwinia herbicola group bacteria was performed by API bacterial population isolated from mulberry leaves in 1993 20E identification kit and acid production from some carbohydrates which was assayed by incubating in un- shaken aqueous solution (1% carbohydrates, 1% pep- tone, with bromocresol purple as an indicator) for 7 days. Identification of Pseudomonas syringae pv. mori was done by the simple identification methods, such as an agglutination reaction to the antiserum of P. syringae pv. mori S6807 (MAFF 810007), a fluorescein produc- tion on King's medium B and pathogenicity to mulberry as previously reported by Sato et al.15)

a) NT: Not tested.

Fig. 1. Survival of Pseudomonas syringae and Fig. 2. Survival of Pseudomonas syringae and

Erwinia herbicola group bacteria on mul- Erwinia herbicola group bacteria in mul-

berry leaves. Bacterial numbers indicate berry pyralid (Glyphodes pyloalid). Bacte-

cfu per gram of mulberry leaves (fresh rial numbers indicate cfu per one larva.

weight). Date indicate when mulberry Date indicate when larvae were sampled. • :

leaves were sampled. • : Erwinia herbicola Erwinia herbicola group bacteria, •¡:

group bacteria, •¡:Pseudomonas syringae. Pseudomonas syringae. Ann. Phytopathol. Soc. Jpn. 61 (5). October, 1995 441

unidentified bacteria were also isolated from all sam- Yellow pigmented strains, TM2 and Mei 7, were ples, the population of the each bacteria was much lower thought to be Erwinia herbicola group bacteria from the than those of E. herbicola group bacteria and of P. results of API 20E identification kit (profile indexes of syringae. TM2 and Mei 7 are 1244573). Moreover, both strains

Proportion of INA bacteria among total epiphytic produced acid from carbohydrates such as inositol, bacteria from mulberry leaves are shown in Table 1. raffinose, cellobiose and glycerol. E. herbicola strains INA bacteria were detected from all samples collected used as a control did not produce acid from these carbo- during October to November, showing that 48.1% of P. hydrates. Accordingly, TM2 and Mei 7 were identified syringae strains and a few of E. herbicola group bacteria as Erwinia ananas. have an ability of ice nucleation activity. A survey of acid production from these carbohydrates Survival of INA bacteria in mulberry pyralid in E. herbicola group strains including non-INA bacteria

(Glyphodes pyloalis) and other organisms indicates that 58% of the strains (7/12) isolated from the Population of bacteria surviving in the insect Glypho- mulberry trees and 75% of the strains (6/8) isolated des pyloalis was surveyed in the samples collected from from the insect were E. ananas. Other strains were E. mulberry fields during October to November in 1993. E. herbicola or undetermined strains. herbicola group bacteria were detected as the dominant Relationship of frost injury and population of flora in most larvae (19/22), but P. syringae was detect- INA bacteria in mulberry trees able from only 6 samples out of 22 larvae (Fig. 2). There was a frost inducing necrosis in most mulberry Proportion of INA bacteria among total bacteria leaves in NISES field on November 25th, 1993. Popula- from mulberry pyralid are shown in Table 2. Three tion of INA bacteria isolated from frost-damaged leaves strains of P. syringae and one strains of E. herbicola with necrotic spots and undamaged green leaves was group bacteria expressed their ice nucleation activity. investigated. These leaves were randomly collected at a Intensity of INA of these strains was investigated by frost morning (-1.7•Ž). Bacteria were isolated by homog- freezing temperature and time required for freezing. All enizing method (sample 1 to 7) or shaking method (sam- the strain did not freeze at higher than -3.5•Ž after 5 ple 8, 9). All samples contained a high population (104 to min. These strains began to freeze -4.0•Ž after 5min, 107 cfu per gram of leaves) of the bacteria, P. syringae -4 .5•Ž after 2-4min, -5•Ž and -6•Ž after 1min. and E. herbicola. Nine shoots were sampled from nine INA P. syringae and E. herbicola group bacteria were locations in the field. In most shoots (7/9), no difference isolated also from a weed, golden rod (Solidago altissima of INA bacteria population between necrotic leaves and

Japanese name=seitaka-awadachisou) in the same green ones was observed (Table 3). In two shoots (sam- mulberry field and INA E. herbicola group bacteria were ple 8, 9), however, a clear difference was observed in the obtained from rice plants in the paddy field next to the population of INA P. syringae pv. mori between both mulberry field (Table 2). types of leaves. Identification of INA bacteria Some un-budding or abnormal budding shoots which

All strains of Pseudomonas-type colony, including the were distinguishable from twig blight disease and other strains isolated from the insects, produced a fluorescein diseases were observed in the next spring (May, 1994). on King's medium B, and showed a positive aggluti- Necrosis was also observed in all basic tissues of un- nation-reaction to antiserum of P. syringae pv. mori budding winter buds after cutting by a razor blade. INA S680715). Moreover, three strains, TM5 from mulberry, bacteria of P. syringae but not INA E. herbicola group Mei 40 and Mei 44 from the insect, selected randomly bacteria were always detected at a high population of represent weak pathogenicity to mulberry. From the 104 to 107 from the necrosis parts of buds, showing 40 to foregoing results, these strains were identified as P. 100% of population proportion. Moreover, abnormal syringae pv. mori. necrosis-buds appeared just after budding had also a high population of INA P. syringae pv. mori.

Table 2. Proportion of ice nucleation-active (INA) bacterial population isolated from mulberry pyralid and a weed, golden rod (Solidago altissima) collected from mulberry fields, and Table 3. Proportion of ice nucleation-active bacteria rice plants in a paddy field being adjoin to the isolated from frost-damaged or undamaged

mulberry field leaves collected in a frost morning (-1.7•Ž)

on Nov. 25, 1993

a) No. of INA bacteria/no. of tested bacteria 442 日本植物病理学会報 第61巻 第5号 平成7年10月

mulberry trees was first described by Takahashi et al.19) DISCUSSION They suggested that a frost (-3.3•Ž) occurred on November 4th in 1981 induced frost formation and The survival of INA bacteria in mulberry trees was necrosis formation in winter buds mediated by INA first described by Takahashi et al.16,18,19)They showed bacteria and induced resulting un-budding in next spring that population proportion of INA P. syringae pv. mori from the following facts: 1) a high population of INA P. were 32.3% and 44.8% among total P. syringae isolated syringae survived in all un-budding parts, 2) November from mulberry leaves and buds, respectively. Similar is when plants have not still acquired their freeze- population proportion of INA P. syringae pv. mori was tolerance ability. Similar results supporting this hypoth- observed in the present study. This is interesting result esis were obtained in the present study. All un-budding from the view of population change of INA bacterial parts or abnormal necrosis-buds appeared just after flora during about 10 years, since both works were budding in next spring (May) contained a high popula- conducted by the similar method and in the same field of tion of INA P. syringae, but not INA E. herbicola, NISES. Although Takahashi et al.16,18,19)did not describe suggesting that INA bacteria associated with frost on INA E. herbicola group bacteria, we found a new injury may be P. syringae pv. mori not but E. herbicola- member of INA bacteria surviving in mulberry trees. group bacteria. This bacterium was identified as E. ananas, although its However, an apparent relationship between appear- population proportion among total E. herbicola group ance of necrotic spots or parts in mulberry leaves in- bacteria was much lower than that of P. syringae pv. duced just after a frost falling (-1.8•Ž) on November mori. Whereas INA E. herbicola group bacteria survived 25th in 1993 and population of INA bacteria was not on other organisms such as a weed, Solidago altissima shown. Although samples 8 and 9 obtained by a shaking and rice plants at a relatively high population propor- method (Table 2) represented a clear differences tion. Bacterial population on mulberry leaves trended to between damaged and un-damaged leaves, but it was not increase in November (Fig. 1) and it may depend on observed in the samples collected after a frost (-2.5•Ž) physiological state of leaves (older aged leaves) and at November 16, 1994 (data not shown). other climatic factors. Two INA bacterial species, P. syringae and E. ananas, We thank Dr. K. Takahashi, Research Inst. Japan Plant. were detected from the insect, mulberry pyralid. To our Protect. Assoc., and Dr. A. Shirata, Natl. Inst. Seric. & knowledge, these bacteria are the first INA bacterial Entomol. Sci., for their useful advices. species detected in insects although some species of INA bacteria and fungi from other insects have been Literature cited reported6,7,9,20). E. herbicola with ice nucleation ability has been isolated from the pupae of diamondback , 1. Arny, D.C., Lindow, S.E. and Upper, C.D. (1976). Frost Plutella xylostella6,7) and INA Enterobacter agglomerans sensitivity of Zea mays increased by application of and Enterobacter tayloae were isolated from the insects, Pseudomonas syringae. Nature (London) 262: 282-284. Ceratoma trifurcata and Hippodamia convergens9). E. 2. Beji, A., Mergaert, J., Gavini, F., Izard, D., Kersters, K., agglomerans is a member of a large, heterogeneous Leclerc, H. and DeLey, J. (1988). Subjective synonymy group of bacteria in the E. herbicola-E. agglomerans of Erwinia herbicola, Erwinia milletae, and Enterobacter complex2). So the difference of species between this E. agglomerans and redefinition of a taxon by genotypic agglomerans strain and our E. ananas strain is not clear. and phenotypic data. Intl. J. Syst. Bacteriol. 38: 77-88. Whether or not these INA bacteria isolated by us or Lee 3. Goto, M., Goto, T. and Inaba, T. (1989). Identification et al.9) originated from those multiplied on insect organs of ice nucleation-active bacteria isolated from frost- including guts is still uncertain. Since the homogenized damaged vegetable leaves. Ann. Phytopathol. Soc. Jpn. insect samples include guts containing mulberry leaves 55: 330-335. or other plants after feeding, probably a part of INA 4. Goto, M., Huang, B.L., Makino, T., Goto, T. and Inaba, strains, especially of P. syringae, may be originated from T. (1988). A taxonomic study on ice nucleation-active feeding leaves. 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