BIOLOGICAL CONTROL OF THE CROWN GALL DISEASE

M. N. Schroth, A. H. McCain and J. Harrison

ABSTRACT

A combination of the crown gall biocontrol strain K-84 and anotherantagonist JH-l was better than K-84 alone in controlling someAgrobacterium strains. Strain K-84 surprisingly controlled someresistant Agrobacterium strains when heavy concentrations of K-84were used. Strain K-84 could control sensitive strains of Agro-bacterium up to 48 hr. after wounds were inoculated with the path-ogen at temperatures of 70 F and below. The ability of K-84 tocontrol various strains of Agrobacterium can only be tested bychallenge inoculation on the rootstock of interest.

OBJECTIVE

1. To find new biocontrol strains that will inhibit the strainsof agrobacteria that are resistant to K-84, the currentlyused biocontrol strain.

2. Develop an inoculum of biocontrol strains that will protectwalnuts and prunes from the different strains of agrobacteriathat occur in California soils.

3. Test ability of K-84 to control resistant strains on specificrootstock using heavy concentrations.

4. Determine affect of temperature on crown gall initiation andability of K-84 to control the disease. How soon must woundedtissues infested with the pathogen be treated with K-84 toprevent infection.

PROCEDURES

1. Bacteria from soils suspected or known to be suppressive tothe crown gall disease are isolated and tested as possiblebiocontrol agents.

2. The tests are done by wounding walnuts and indicator plants,swabbing the wounds with a soil suspension, and then isolatingfor bacteria that have the ability to colonize the wound site,but not cause galls. Bateria with this ability have thecharacteristics necessary to compete with crown gall bacteria.

3. Bacteria isolated from wounds are tested in vitro for antibioticproduction against crown gall bacteria.-The positive strainsare then tested by coinoculating plants with crown gallbacteria.

4. Field and greenhouse tests are made with potential biocontrolstrains.

5. Coinoculate using resistant strains with increasing dosagesof K-84. Use tomato and sunflower as indicator plants.

6. Inoculate indicator plants with pathogen followed by treatmentwith K-84 at intervals up to 48 hr. later. This work willbe done at different temperatures.

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RESULTS

An assortment of bacteria were found that were as effective asK-84 in controlling the crown gall disease. Some of these bacteriacontrolled resistant strains of Agrobacterium. However, in subse-quent experlments it was discovered that it was particularly impor-tant to consider time of application of the biocontrol agent inrelation to when the wound (site for infection) occurred. Althoughvarious bacteria were as good or better than K-84 when the woundwas coinoculated with the pathogen and biocontrol agent simulta-neously, only K-84 was effective upon delayed treatment such aswhen the pathogen was inoculated 12-24 hr. in advance of the biocon-trol agent. This can be seen by inspecting Tables 1 and 2. Thestrain JH-l is an example of one of the biocontrol strains thatwas compared with K-84. Tables land 2 are examples of many experi-ments and show some of the facets of our experimentation.

Tomato and sunflowers were used as indicator plants to test theability of K-84 to control resistant stralns of the crown gallpathogen. Surprisingly, some of the resistant ,strains were con-trolled alsmost as well as susceptible strains, but this dependedon the host. It was also discovered that the ratio of K-84 inoculumto pathogen was highly important. Table 1 presents examples ofthis using a representatlve sample of the strains that we usedin the tests. Note that resistant straln A-20 was controlledon tomato as well as the susceptible strain C-48. However, otherresistant strains were not controlled on tomato. On the otherhand with a different host, sunflower, note that all of the resis-tant strains were controlled.

In some cases, a mlxture of K-84 and JH-l gave the best control.Since JH-l was inhibitory to K-84, K-84 was made reslstant toJH-l so that the comblned inoculum would be compatible 0

Table 2 shows ~he importance of timing in treating infection siteswith K-84. ThlS is particularly important Slnce most growersdo not immediately treat trees upon harvesting them from the soil.This experiment shows that K-84 effectively controls a sensitivestrain (25-1:6) of Agrobacterlum even if the treatment is applied24 hr. after lnocul ation with the pathogen but only at 70 F andlower.

In subsequent experiments, the temperature of inoculation provedto be the governing factor which determines whether or not K-84will be effective in delayed treatments up to 48 hr. At highertemperatures (above 70F), delayed treatments did not work.

CONCLUSIONS

1. Many resistant strains of Agrobacterium tumefaciens can becontrolled by the biocontrol strain K-84, but it may requireheavy concentrations to effect this.

2. The ability of K-84 to control resistant strains depends onthe host. Indlcator stralns cannot be used to determine this.Therefore, lnoculations, in the case of walnut, and prune,must be done with the rootstock of interest.

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3. Although various bacterial strains were discovered that wereantagonistic to crown gall strains, they did not compare withK-84 when the biocontrols were appli~d at given times afterinoculation with the pathogen.

4. K-84 must be applied almost immediately when plants are woundedwhen the temperature is above 70 F and if the pathogen ispresent. Otherwise, treatment up to 48 hr. later will resultin an ineffective biocontrol.

5. A combination of K-84 with another bacterial antagonist wasmore effective than K-84 alone in controlling some Agrobacteriumstrains.

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Table 1. Treatment of wounds with K-84, JH-1 or a combination of both, and

subsequent inoculation with a representative number of crown gallstrains including those that are resistant to the antibiotic of K-84

* 1:1 and 1000:1 is 10~ and 10H colony forming units, respectively, of K-84 to105 of the pathogen.

Table 2. Effect of temperature on ability of K-84 to control crown gallwhen treatments of K-84 are made 24 hr. after the wounding ofplants.

Tomato host

strain 25-1:6Number of galls out of 36 inoculations

70 F 80 F

K-84 treatment

Check136

2034

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.Numberof galls out of 20 inoculationsTomato Host K-84 K-84. JH-1 K-84 & JH-1Strain sensitive 1:1* 100:1 1000:1 1:1 100:1 1000:1 1000:1 Check

25-1:6 + 7 a a 9 a 2 a 14C58 + 20 a a 20 20 11 a 20A20 - 19 13 2 20 15 9 a 20Maz 2-3 - 19 15 9 17 a 2 1 18A49 - 19 14 10 20 15 7 6 19Rasp 3 - 19 19 13 15 4 9 3 20B6 - - 14 - - 8 - 3 20

Sunflower Number of galls out of 40 inoculationsHost K-84 JH-l K-84 & ,JH-l

Strain 1:1 100:1 1000:1 1:1 100:1 1000:1 1000:1 Check

25-1:6 14 a -- a 26 18 a 0 35C58 7 a a 13 9 0 0 26A20 22 1 a 19 8 9 0 37Maz 2-3 17 3 1 9 1 2 1 27A49 30 4 3 - 4 1 a 26Rasp 3 33 16 a 5 6 0 3 29B6 24 2 - 14 5 - 3 35