ROBERTS, PATJLIXE (1974). J . uppl. Bnct. 37, 353-358.

Erwinia rhupontici (Millard) Burkholder Associated with Pink Grain of Wheat

PAULINE ROBERTS

Miwistry of Agriculture, Fisheries and Food, Plant Pathology Laboratory, Hatching Green, Harpenden, Herts., England

(Received 15 January 1974 and accepted 6 May 1974)

SUMMARY. Bactoria isolated from pink-colourcd grains of English wheat were identificd as E'rwiniu rhapontici, and were identical with isolates made by Luisotti & ltapilly (1967) from wheat with a similar disorder in France. Pink grain was reproduced with English and French isolates and with authentic culturos of R. rhnpontici when dovcloping grain of wheat plants was inoculatod, but only after wounding.

IN ENULAND consignments of wheat are occasionally found to contain a low percentage of pink-coloured grains (A. V. Hart, pers. comm.). In samples received a t this labora- tory, the pink colour extended throughout such grains and the endosperm was slightly softer than usual, but otherwise no difference from healthy grains was noticed. Bacteria which produced a pink diffusible pigment on sucrose-peptone agar were always isolated from the pink grains, but Fusarium roseuin which can cause a pink discoloration of wheat grains (Luisett,i & Rapilly, 1967) was never isolated. A similar but more widespread disorder of wheat in France has been described by Luisetti & Rapilly (1967) who isolated a bacterium which produced a pink diffusible pigment in culturc inedia with some of the characters of the genera Enterobacter, Serratia and Pectobacterium. They reproduced the disorder experimentally with this bacterium. In this paper a comparison of the French and English isolates is made, and the bacteria are identified as Emoinia rlmpontici, a pathogen associated until now only with crown rot of rhubarb.

Materials and Methods

Bacterial cultures Bacteria were isolated from pink wheat grains by grinding a small portion of the endosperm in a drop of sterile water and streaking it on to sucrose-peptone agar (SPA) containing: sucrose 20 g; Oxoid bacteriological peptone, 5.0 g; K,HPO,, 0.5 g; MgSO,, 7H,O, 0.25 g ; Oxoid agar No. 3, 12 g ; distilled water, 1 1; adjusted to pH 7.2 and autoclaved at 121' for 15 min. The dominant isolate was a bacterium which produced in 2-3 days at 27" mucoid pink colonies from which the pigment diffused into the agar.

Two French isolates kindly supplied by Monsieur J. Luisetti (NCPPB* 2648, 2649)

* National Collection of Plant Pathogenic Bactoria, Plant Pathology Laboratory, Hatching Green, Harpendon, Horts., England.

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Pauline Roberts

and 5 English isolates from 2 diRerent wheat samples (NCPPB 2558, 2559, 2560; A425/L, A2) were used in this work. Four strains of E'. rhupontici from rhubarb (NCPPB 139, 1578, 1739, 2506) were included in the tests for comparison.

Cultural and biochemical tests The characters studied were thosc recommended by Lelliott (1974) for distinguishing Erwinia spp. Unless otherwise stated, all cultures were incubated a t 27".

O/F twt. The method of Hugh & Leifson (1953) was used and the results recorded after 3 days.

Oxidme and catnlase activity. Cultures grown on nutrient agar for 2 days were tested for production of oxidase (KovAcs, 1956) and catalase (Cowan & Steel, 1966).

Utilization of organic acids. The method of Dye (1968) was followed. Amino acid decarboxyluses. The method of Mdler (1955) was used. Urease. Urease medium (Christensen, 1946) was inoculated and results read after

Growth at 36". Cultures were incubated for up to 7 days in a waterbath a t 36 & 0.2". Cluconate oxidation. Cultures in Shaw & Clarke's medium (1955) were incubated on

a shaker for 4 days a t room tempcrature; a positive reaction was the production of an orange/brown colour after the culture, mixed with an equal volume of Benedict qualitative reagent, had been held a t 100" for 10 min.

Reducing compounds from sucrose. A 2-day shake culture grown in sucrose broth (sucrose, 4 g ; peptone, 1 g, beef extract, 0.5 g; distilled water, 1 1) was tested as described under gluconate oxidation.

7 days' incubation.

Nitrate reduction. The method outlined by Spencer (1969) was followed. Methyl red and Voges-Proskauer (acetoin) reactions. Cultures were tested after

2 and 5 days according to the methods of Cowan & Steel (1966), using O'Meara's (1931) method to detect acetoin.

Gelatin liquefaction. Stab cultures in Oxoid Nutrient Gelatin were incubated for 21 days.

Phenylalanine deamimse. Cultures grown on phenylalanine agar for 2 days were tested with 10% (w/v) aqueous FeC1, (Report, 1958).

Indole production. KovScs (1928) reagent was added to peptone water cultures which had been shaken a t room temperature for 2 days.

Casein hydrolysis. The milk agar of Dye (1968) was used. Growth in 5% NaCl. Cultures were shaken a t room temperature for 5 days in Oxoid

Nutrient Broth adjusted to a concentration of 5% (w/v) NaCl. Lecithinase. Cultures were grown on Oxoid Egg Yolk Agar for 7 days. Phospluctase. The methods of Cowan & Steel (1966) were followed, but cultures

Pectate liquefaction. The medium of Paton (1959) was used. Acid from carbohydrates. The basal medium was 1% (w/v) peptonef 1 ml of a 1%

(w/v) alcoholic solution of bromocresol purple, adjusted to pH 7.2 and autoclaved a t 121 O for 15 min; to the sterile base, a filter-sterilized solution of the carbohydrate was added to a final concentration of 1% (v/v). Cultures wore examined for acid produc- tion for up to 28 days.

were grown on 0 . 0 5 ~ o (w/v) sodium phenolphthalein diphosphate agar for 2 days.

Erwinia rhapontici associated with wheat 355

Action on potato, onion and c z ~ c u d ~ e r slices. Potatoes and cucumbers were surface sterilized by flaming them briefly in alcohol, peeled aseptically and cut into slices c. fimm thick. Each slice was transferred immediately to a sterilc Pctri dish con- taining water (c. 2 Inm deep). Onions, their outer scale leaves removed, were flamed in alcohol and sliced in the same way. Slices were stab inoculated 11 itli bacteria from a young nutrient agar culture, moistened with water and incubated for 3 days.

Pathogenicity tests Growth from a 2-day nutrient agar culture was suspended in sterile distilled water to a concentratioii of c. lo9 cells/ml. Wheat cv. Cappelle Desprez at the niilky grain stage was inoculated in July by dropping the bacterial suspension on to the ears. In one set of inoculations the grains were wountlcd by stabbing with a needle; in another set the grains were not wounded. The r i p grain was haiwstrd one month after inoculation.

TABLE 1

Some biocheniiml chnructers of Erwinia rhapontici and bacteria isolated f rom pink grain of wheat

English pink French pink Biochemical test grain isolates ( 5 * ) grain isolates (2) E. rhapontici (4)

- - Urease - Growth a t 36" + + + t Oluconate oxidation + + + + Reducing compounds from sucrose - Nitrate reduction + + +

d t Methyl red - Voges-Proskauer + + d t Gelatin liquefaction - Phenylalanine deaminaqe - Indole production - Casein hydrolysis - Growth in 5% NaCl + + + Lecithinase - Pectate liquefaction - Pink pigment (on SPA) + + +

dS Phosphatase -

-

-

- - - - - - - -

- - - -

-

+, Positive result; -, negative result; tl, difforont isolatcs gave diffcrent results. * Number of isolates studied. t Differs from data given by Dye (1969). 1 Differs from data given by Graham (1972).

Results

Cultural and biochemical tests All the isolates from pinli grain of wheat and all the E . rhpontici strains were motile, Gram negative rods which were oxidase negative, catalase positive and which used glucose fermentatively without production of gas. All isolates uscd formate, malonate, tartrate, lactate, citrate and galacturonate, but none produced dccarboxylases for

Pauline Roberts

L-lysine, L-arginine, L-orthinine or L-glutamate. Other biochemical characteristics are in Table 1, and details of acid productioii from carbohydrate are in Table 2. Although no isolate liquefied the pectate medium, all isolates cnuscd a soft rot of potato, onion and cucumber slices, with a noticeable pink colour on potato and onion. All isolates produced a pink diffusible pigment on SPA.

T A s L E 2 Acid production from carbohydrates by Erwinia rhapontici and

bacteria isolated from pink grain of wheat

English pink Prench pink Carbohytlratu grain isolutos (a)* gruin isolatos (2) 13. rhrrpontici (4)

a-Mothyl glucosido Xylose Raffinoso Ihlcitol Inositol Lactoso Melezitose Sucrose Arabinoao

- + + + + + +

d

+ + + + + + +

d t

+ , Positive rosult; - , nogativo rosdt , ; ti, tlifforont isohto3 gave tliCfororent rcsults. * Number of isolatos stutliod. t Diffurs from clutn givon by Dyo (19G9).

TABLE 3 Results of pathogenicity tests on wheat G I ) . Cappelle Desprez

Incidence of pink grains Inooulum .A 3

Woundod grains Unwoundod grains

Ihglisli pink grain isolatos 4/49 0/30 E. rhnpontici 3/44 0/19 H,O Control 0127 0/10

l'uthogeiaicity tests Results of pathogcnicity tests on wheat cv. Cappelle Desprez are summarized in Table 3. The same pink bacterium was re-isolated on SPA from all the pink grains, usually as the dominant organism, but i t was not found in normal coloured grains.

Discussion

The biochemical characters of the French and English isolatcs from pink grain of wheat were identical and were very similar to those of the authentic E. rhapontici strains (the differences being in tlie production of reducing compounds from sucrose alld of acid from dulcitol and melezitose). When E . rhnpontici strains and the English

Erwinia rhapontici associated with wheat 357

pink grain isolates were inoculated on to wheat ears, in both cases similar small proportions of pink grains were later found in the harvested wheat. These results are good evidence that the bacteria isolated from pink grain of wheat are Emoinia rhapont ici.

I n this work, results of the authentic E. rhapontici strains differed from the data given for the species by Dye (1969) and quoted in Bergey’s Manual 8th Edition (Lelliott, 1974) in the following : tartrate and galacturonate utilization, growth at 36” and methyl red test (all negative according to Dye); Voges-Proskauer reaction and acid from xylose (both positive according to Dye) and phosphatase (positive according to Graham, 1972). Ewinia rhapontici has also been reported not to cause a soft rot of potatoes (Metcalfe, 1940)) though in this work all strains did so.

Only a small percentage of naturally infected pink grain was found in thc English wheat samples. In the pathogenicity tests on wheat cv. Cappelle Desprez, where infection conditions may not have been optimal, the English isolates from pink grain infected only c. 8% of wounded grains, as did the authentic E. rhapontici strains; unwounded wheat grains were never affected. However, inoculation of French isolates into wounded grains of wheat cv. Moisson by Luisetti & Rapilly (1967) reportedly caused development of numerous pink grains. From thcse results, it seems likely that the introduction of the bacteria into a wound is a prerequisite of infection: Luisetti & Rapilly (1967) noted that the rather severe outbreak in France of wheat pink grain in 1966 coincided with an infestation ofgall midges (Cecidomyidae), and crown rot of rhubarb by E. rhpontici is a wound infection often associated with eelworm infestation (Metcalfe, 1940). The status of E. rhapontici as a plant pathogen has not been well-established and it may be an opportunist pathogen which perhaps is primarily soil inhabiting, only infecting a plant when conditions are especially favourable.

In the UK pink grain of wheat is of no economic importance, but confusion might arise because of the superficial likeness of pink grain associated with E. rhapontici to the pink colour of some organo-mercury seed dressings. When grain is to be used for flour milling it is important to establish that the pink colour is not caused by a mercury- containing compound.

I should like to thank Mr A. V. Hart of the Flour Milling and Baking Research Association for drawing my attention to pink grain of wheat and supplying the samples, and Mrs J. Sellwood for her help with the experimental work.

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