Journal of Applied Bacteriologv 1976, 41, 277-281

Filter Mud as a Carrier for Rhizobium lnoculants

HELEN PHILPOTTS

Agricultural Research Centre, Wollongbar, New South Wales 2480, Australia

Received 29 March 1976 and accepted 22 April 1976

Laboratory tests indicated that fdter mud, a waste product from sugar cane mills, proved to be a suitable carrier for Rhizobiurn inoculants, provided it was not dried by heating. Autoclaving air dried material was not detrimental and ensured high survival when held at room temperature. The adverse effect of oven drying, which was increased by subsequent autoclaving or gamma irradiation, lessened with time.

FINELY GROUND PEAT is widely used for Rhizobium cultures for inoculating legume seed, as in this form the cultures are easily distributed, and survival of the rhizobia on inoculated seed is better than when agar cultures are used (Date 1970). However, peat deposits may not be available or the quality of the peat may not be suitable.

Alternative carriers of uniform composition would, therefore, be of considerable value. One material which shows promise is filter mud, a waste product from sugar cane mills. Filter mud is the finest deposit obtained during the filtration and clarification process of crushed cane. It consists of very fine cane fibre particles and silt in a ratio of approximately 0.6 : 1. Machine harvesting of the cane results in lower ratios, 0.5 : 1 being more typical. Table 1 shows the composition of filter mud from cane harvested by hand during the 1974 season from a mill in northern New South Wales. It has a very high moisture holding capacity (400% moisture at field capacity). The pH value is usually close to 7 and is controlled by the addition of lime to the liquor. It may be in- creased to about 8 if an interruption to processing is anticipated.

TABLE 1

Average composition of filter mud for the 1974 season at Broadwater mill New South Wales

A , ignition I A Major constituents (%) Loss on Mineral analysis (% dry matter)

‘Mud Fibre Sugars Water (%dry P,O, CaO MgO N K,O Minor*’ solids matter) elements

11.6 7.8 1.6 78.8 81 3.91 0.99 1.23 1.22 0.11 0.025 (9-15)t (6-10) (1-3) (75-78)

* Cu, Mn, Zn, Bo. t Range. Data supplied by Colonial Sugar Refining Co. Ltd, Broadwater.

[2771

278 H. PHILPOTTS

Materials and Methods

Inoculant preparation

Samples of filter mud were collected from the mill at different times during two crushing seasons. After drying and grinding to pass through a 0.75-mm mesh, in- oculants were prepared using TAl and CB756, the current Australian commercial Rhizobiurn strains for clover and cowpea, respectively. The inoculants were prepared by adding broth to carrier in a ratio of 1 :2 (v/w). As water was added to the dry material ( 1 : 2 v/w) before autoclaving, an equal quantity of sterile water was added to the broth used for inoculating the filter mud which was not autoclaved. This kept the ratio of broth to carrier constant and the extra moisture was found more satisfactory. At least two replicates were prepared.

The number of viable rhizobia in the inoculants was determined by the plant infection method (Vincent 1970) using duplicate tubes and 10-fold dilutions. The first count was made after 2 weeks‘ incubation at 28 O C . The first sample of filter mud (pH 7.1) was air dried for 9 days and inoculants prepared using unsterilized and autoclaved material. Rhizobiurn counts were made after 0, 4 and 8 weeks’ storage at 8 O C . The second sample (pH 7.0) was oven dried at 100°C for 4 days to speed up the drying process. Inoculants were prepared using unsterilized, autoclaved and gamma-irradiated material. Additional inoculants of TA 1 were prepared, from unsterilized and irradiated material of Sample 2 , after 16 weeks’ storage of the former and 8 and 16 weeks of the latter. Numbers in the inoculants were determined after holding at 8 O C for different periods.

A third filter mud sample (pH 8.1) was either air or oven dried and from each lot in- oculants were prepared from unsterilized or autoclaved material. Rhizobium counts were made after 0 and 12 weeks’ storage at room temperature (c. 2OOC).

Seed inoculation

Inoculants, prepared from unsterilized filter mud of Sample 1, were used to inoculate seed of white clover (Trifolium repens) and glycine (Glycine wightii). One gram in- oculant in 2 ml gum arabic (30% w/v solution) was used for 35 g of white clover and 70 g of glycine seed. A coating of fine lime was applied. The number of viable rhizobialseed, on duplicate samples, was determined after 5 days, 5 and 10 weeks, using the plant infection method.

Results and Discussion

Table 2(a) shows that rhizobium numbers in the inoculants prepared from the air dried filter mud (Sample 1) whether sterilized or not, were high, and similar to the standard set by the Australian Inoculant Research and Control Service for commercial peat in- oculants ( 1 x 109/g carrier) (Roughley 1975). Numbers were maintained over the holding period of 8 weeks.

Numbers in the inoculants prepared from oven-dried filter mud (Sample 2) were con- sistently lower than those of Sample 1. For both strains autoclaved inoculants were in- ferior to unsterilized and the irradiated ones were failures [Table 2(b)l. TAl was

FILTER MUD FOR RHIZOBIUM INOCULANTS 279

TABLE 2 (a) (b) (c) Growth and survival of rhizobia in filter mud previously treated in diflerent ways

Log,, no. rhizobiaig inoculant after holding at 8 OC for weeks*

Filter mud sample and Rhizobium h I > strain 0 4 8 12 36 treatment

(a) Sample I (air dried) Unsterile

Autoclaved

(b) Sample 2 (oven dried) Unsterile

Autoclaved

Irradiated

(c) Sample 2 (oven dried

16 wk after drying and stored)

unsterile irradiated 8 wk previously peat control

24 wk after drying irradiated 16 wks

previously peat control

TA 1 CB756 TA 1 CB756

TA 1 CB756 TA 1 CB756 TA 1 CB756

TA 1 only

>8.95 2 8 . 2 3 28 .95 28 .23

6 .26 7.73

< 4.76 5.50

< 4 . 7 6 t < 2.46t

7.26 <2.73

7.26

8.05 8.26

>9,26 8 .23 8.84 9.02

6.77 8.00

<2 ,76 5 .84

<2.76 <2.46

10.65 8.42 8.65 9.42

6.55 7.57 6.42

<1.76 5.55 6.77

<1.76 (1.76 7.02

8.75 8.55 4.95 7.52 8.45 8.55

~ ~ ~~~

* Time after 2 weeks maturing following inoculation. t Repeat preparations had < 1.76.

suppressed in the autoclaved or irradiated filer mud more than CB756, whose numbers increased in the inoculants after holding for 36 weeks. In the unsterilized material, however, TA 1 numbers were maintained, whereas CB756 numbers declined between 12 and 36 weeks [Table 2(b)l.

The restricting factor in Sample 2 diminished with time, as shown in Table 2(c). Six- teen weeks after oven drying, numbers in inoculants of unsterilized filter mud were the same as in peat controls. The irradiated material still inhibited growth, but it had been irradiated 8 weeks after drying. Sixteen weeks after irradiation there was no adverse effect. Although the inoculants prepared with filter mud irradiated 8 weeks previously had very low initial numbers, these increased over a holding period of 12 weeks [Table

Drying the filter mud at 100 O C was suspected of being the cause of the poor results with Sample 2, since Roughley (1970) found this had an adverse effect on peat. Oven dried inoculants of filter mud prepared from Sample 3 were inferior to air dried ones, thus confirming the adverse effect of heat (Table 3).

Low pH value was considered a possible cause of increased inhibition of growth after autoclaving oven dried filter mud, as these treatments caused a fall of approximately 2 pH units, as compared with 1.2 for air dried, autoclaved material. This would have reduced Sample 2 to pH 5 which is sufficiently low to restrict growth of R . trifolii (Jensen 1942). However, although the pH value of Sample 3 (pH 8.1 when fresh) did

WI.

280 H. PHILPOTTS

TABLE 3 Growth of rhizobia in filter mud (Sample 3), previously treated in diflerent ways, and

survival when held at room temperature

Log,, no. rhizobiaig inoculant

TA I CB756 h

I >

& * Filter mud treatment PH O* 12 wk* 0 12 wk

Air dried Unsterilized Autoclaved

Oven dried Unsterilized Autoclaved

7 . 6 7 .23 < 5 , 7 6 8.55 8.55 6 . 9 8 . 8 4 8.25 9.05 9 .02

Peat control 6 . 4 8 .17 8 .14 8.55 8 .54 * Time after 2 weeks maturing following inoculation.

not fall below 6.2, growth was again suppressed in oven-dried inoculants. Hence, low pH value is not considered a major factor.

Although the initial numbers of strain CB756 were low for the autoclaved oven dried inoculants, after holding for 12 weeks at room temperature the numbers had increased by more than 1.2 log units. No such increase occurred in inoculants of unsterilized, oven dried material, or for TA 1 in either the unsterilized or autoclaved oven dried ones (Table 3). It did occur, however, with TAl in stored irradiated material [Table 2(c)l. The high nutrient content of filter mud (Table 1) would allow multiplication of any surviving rhizobia, once the inhibiting effect disappeared, during storage. In unsterilized material competition from other organisms could suppress this.

Table 3 shows the air dried, autoclaved inoculants of both strains had higher initial numbers than the peat controls and had similar or higher numbers after 12 weeks at room temperature.

The satisfactory results with strain CB756 in unsterilized air dried filter mud were surprising, as this slow-growing rhizobium usually gives poor results in non-sterile peat (Roughley 1970). As found with Sample 2, TA 1 was more susceptible than CB756 to the adverse effect of oven drying and in unsterilized, air dried filter mud the numbers declined during storage at room temperature. The effect of irradiation on air dried material has not been determined but, if detrimental, a period of storage would rectify this.

TABLE 4 The number of rhizobia obtained by inoculating seed with filter mud inoculants,

and their survival when stored at 8OC lLog,, no. rhizobiaiseed

after storage for Rhizobium strain I A

and seed species 5 d 5 wk 10 wk >

TA I on Trfolium repens 4 . 2 2 3 .72 4 . 5 4 CB756 on G!wine nighrii 4 . 2 6 3 .72 4 .22

FILTER MUD FOR RHIZOBIUM INOCULANTS 28 1

Seed inoculated with filter mud inoculants had high numbers of rhizobiaiseed and the numbers did not decline when held at 8 OC for 10 weeks (Table 4). The number of bacteria on glycine seed was very close to the estimated number added to the seed (4.26 cf. 4.30). For white clover the numberheed was high but it was lower than the estimated number (4.22 cf. 5.93). However, it has been difficult to achieve the theoreti- cal number on white clover using peat inoculants and a similar order of difference often occurs.

This work shows that filter mud is a suitable carrier for rhizobium inoculants, provided it is not oven dried. As survival in unsterilized material was variable, steriliza- tion would be advisable, but if irradiated, a storage period might be needed.

I wish to thank the Colonial Sugar Refining Co. Ltd for provision of the filter mud and data concerning it, the Australian Meat Research Committee for financial support, and Mr D. Firth and Mr P. O’Sullivan for technical assistance.

References

DATE, R. A. 1970 Microbiological problems in the inoculation and nodulation of legumes. Plant and Soil 32, 703-725.

JENSEN, H. L. 1942 Nitrogen fixation in leguminous plants. I. General characters of root nodule bacteria isolated from species of Medicago and Triflium in Australia. Proceedings of the Linnean Society of New South Wales 67, 98-108.

ROUGHLEY, R. J. 1970 The preparation and use of legume seed inoculants. Plant and Soil 32,

ROUGHLEY, R. J. 1975 In Symbiotic Nitrogen Fixation in Plants ed. Nutman, P. S. Cambridge:

VINCENT, J. M. 1970 A Manual for the Study of Root Nodule Bacteria IBP Handbook No. 15.

675-701.

The University Press.

Oxford and Edinburgh: Blackwell Scientific Publications.