Study on the growth, persistence and interaction between Corynebacterium insidiosum (McCullock) Jensen and Rhizobium meliloti Dang. in soil, and in the infection of Medicago sativa L.

Abstract

Growth and persistence of C. insidiosum and R. meliloti in soil and rhizosphere, and their interactions with F. oxysporum and V. alboatrum in the infection and nodulation of lucerne plants were investigated.

The droplet method, plaque count technique, plant infection technique and the FA technique were used to isolate, identify and count C. insidiosum and R. meliloti in soil. The FA technique was quantitative, sensitive and easy to perform. The plaque count technique was quantitative, its sensitivity depended on the specificity of the bacteriophage used and it required numerous assays for each sampling. The plant infection technique was laborious, time consuming and in general less sensitive than the FA technique and plaque count technique. The droplet method was simple to perform but the lack of selective media for C. insidiosum and R. meliloti rendered it unsuitable for the detection of these bacteria in field soil. Both bacteria could survive for at least 132 d when inoculated into gamma sterilised soil in which persistence wase favoured at soil moisture levels of 58% W.H.C. and 100% W.H.C. and at 9°. In field soil, C. insidiosum persisted for less than 21 d but R. Meliloti was still present after 42 d. C. insidiosum persisted in diseased tissue for more than a year at 52% R.H. but only for 14 d at 100% R.H.

C. insidiosum and R. meliloti were stimulated to multiply in the vicinity of roots of lucerne on agar media. In soil, the growth of the two bacteria was stimulated in the rhizosphere when the inoculum levels were 10⁷ cells ml⁻¹ and 10⁴ cells ml⁻¹, but not at 10¹ cells ml⁻¹.

Infection of lucerne by C. insidiosum was favoured when the host plants were wounded and at the fourth trifoliate leaf stage than younger stages. The number of plants infected was more at temperature of 21° than 30°, 14° and 10°; at soil moisture levels of 58% W.H.C. and 100% W.H.C. than 30% W.H.C.; at high N and low K conditions. Soil pH in the range of 5.1 to 7.8 did not influence infection. Infection was 90% at inoculum level of 10⁷ cells ml⁻¹ , 50% at 10⁶ cells ml⁻¹ and 0% at 10² cells ml⁻¹.

The number of nodules formed by R. meliloti and nodulation percentage were 7.5 per plant and 70% respectively at inoculum levels of 10³ cells ml⁻¹ and 0.1 nodules per plant and 10% nodulation at inoculums level of 10² cells ml⁻¹.

A cross streak test on agar media showed that the growth of C. insidiosum and F. oxysporum or V. alboatrum were affected by the presence of one another. Similar observations were made with R. meliloti and the fungal pathogens. Glasshouse experiments showed that incidence of bacterial wilt infection was lessened when R. meliloti were present in the inoculation mixture with C. insidiosum. At inoculums level of 10⁷ cells ml⁻¹ of C. insidiosum, infection decreased when the concentration of rhizobia in the mixture increased. C. insidiosum, F. oxysporum, and V. alboatrum all caused reductions in nodule number and forage dry weight when inoculated alone. Mixed infections of C. insidiosum with either fungal pathogen caused even further reductions.

Toxin isolated from liquid culture of C. insidiosum inhibited the growth of R. meliloti on an agar medium and reduced the number of nodules formed on lucerne plants.

The FA technique showed that growth of C. insidiosum was less and the pathogen progressed slower in the xylem vessel elements of bacterial wilt resistant variety Saranac than susceptible variety Wairau.