An Improved Method to Evaluate Avocado Rootstocks for Resistance to Phytophthora cinnamomi

Proc. of Second World Avocado Congress 1992 pp. 101-104

An Improved Method to Evaluate Avocado Rootstocks for Resistance to Phytophthora cinnamomi

Marius De V. van der Merwe

Dept Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, Republic of South Africa

Abstract. A detached root technique for evaluation of resistance in avocado root-stocks makes use of zoospores of Phytophthora cinnamomi Rands (P.c.) as inoculum source. The production of zoospores is a lengthy process and requires a precise balance of nutrients. Mycelium fragments of P. cinnamomi were therefore evaluated as an alternative inoculum source. Resistance was determined by measuring lesion length and tissue colonization after 24 and 48 hours. Zoospore and mycelium inoculum compared well as sources of inoculum, both reflecting known field resistance of the various rootstocks. Mycelium of P. cinnamomi can therefore replace zoospores as an inoculum source in the detached root technique.

Root rot caused by Phytophthora cinnamomi Rands is the most serious disease on avocado (Persea americana Mill.) (Zentmyer, 1984). The detached root technique for evaluation of resistance in Persea spp. was first described by Kellam and Cof-fey (1985), Dolan and Coffey (1986) and modified by Botha, Wehner and Kotze (1989). This technique makes use of zoospores of P. cinnamomi as inoculum source. However production of zoospores is a lengthy and laborious process and sporangium production requires a precise balance of certain nutrients (Chen and Zentmyer, 1970; Gisi, Zentmyer and Klure, 1980), and cannot always be guaranteed.

Mycelium of P. cinnamomi have been shown to infect plants (Rodger, 1972; Weste, 1974), and the aim of this study was to compare mycelium fragments and zoospores as sources of inoculum for use in the detached root technique.

Materials and Methods

Roots from 12- to 14-month-old seedlings of P. americana cultivar Edranol (susceptible) (Snyman, et a/., 1984), and vegetatively propagated (Frolich and Platt, 1971) seedlings of P. americana selection. Duke 7 (moderately tolerant) (Coffey, 1987), as well as P. schiedeana Nees selection G755 (tolerant) (Coffey, 1987), were used in this study.

Zoospores were obtained as described by Botha et al. (1989). For the mycelium inoculum, 20 5 mm ²potato dextrose agar discs (PDA) previously colonized by P. cinnamomi were inoculated into 100 mL pea broth prepared as described by Chen and Zentmyer (1970). After incubation at 25C for four days, the fungal mats were homogenized for 30 s with an ultra turrax to produce a mycelial fragment suspension.

Excised root tips (ca. 40 mm in length) from each of the different rootstocks were placed perpendicularly onto two parallel glass rods in petri dishes, containing 1 5 mL water agar in each as described by Botha et al. (1989). Each root tip was inoculated at the region of elongation with either 10 jjL of zoospore suspension containing 7.9 x 10³ per ml motile zoospores or 10 /jL of mycelium ho-mogenate. The root tips were then incubated in the dark at 25C.

Resistance was determined by measuring lesion length after 24 and 48 hours as well as by aseptically cutting the root tips in 4 mm segments after surface disinfecting for 5 s in 70 per cent ethanol. The root segments were then plated out sequentially on a PARPH-medium. After incubation at 25C for three days, the segments from which P. cinnamomi developed were counted and multiplied by four to give the total length of root colonization.

Results

Lesion length after 24 and 48 hours are shown in Tables 1 and 3. Lesion length in the Edranol roots after 24 h was significantly longer than that of Duke 7 and G755 when mycelium was used as inoculum.

No significant differences were detected between the different root-stocks after 24 h when zoospores were used as inoculum. After 48 h the lesion length of Edranol and Duke 7 was significantly longer than that of G755 when mycelium or zoospores were used as inoculum. Tissue colonization is shown in Tables 2 and 4. The same tendency was found in tissue colonization as in lesion length. However, tissue colonization in Duke 7 was greater than in Edranol and G755 after 24 and 48 hours when zoospores were used as inoculum source.

Discussion

The same tendency was obtained between lesion length and tissue colonization when they were used as parameters for determining resistance.

Differences in resistance between the various rootstocks were more obvious after 48 h than after 24 h. Linear colonization of Duke 7 was generally higher than in Edranol and compared well with the findings of Kellam and Coffey (1985), namely that Duke 7 could possibly support higher populations of P. cinnamomi than the susceptible Topa Topa (in this case the susceptible Edranol). Mycelium and zoospores compared well as sources of inoculum, as G755 was significantly less susceptible than Duke 7 and Edranol in both instances. Both types of inoculum also reflected known field resistance of the different rootstocks. Mycelium can therefore be used as a source of inoculum instead of zoospores in the detached root technique.

Literature cited

Botha, T., F.C. Wehner, and J.M. Kotze. 1989. An evaluation of in vitro screening techniques for determining tolerance of avocado rootstocks to Phytophthora cinnamomi. S. A. Avocado Growers' Assn. Yrbk. 12:60-63.

Chen, D.W. and G.A. Zentmyer. 1970. Production of sporangia by Phytophthora cinnamomi in axenic culture. Mycologia 62:397-402.

Coffey, M.D. 1987. A look at current avocado rootstocks. Calif. Grower. 11:15-17.

Dolan, T.E. and M.D. Coffey. 1986. Laboratory screening technique for assessing resistance of four avocado rootstocks to Phytophthora cinnamomi. Plant Disease 70:115-118.

Frolich, E.F. and R.G. Platt. 1971. Use of the etiolation technique in rooting avocado cuttings. Calif. Avocado Soc. Yrbk. 56:97-109.

Gisi, U., G.A. Zentmyer, and L.J. Klure. 1980. Production of sporangia by Phytophthora cinnamomi and P. palmivora in soils at different matric potentials. Phytopathology 70:301-306.

Kellam, M.K. and M.D. Coffey. 1985. Quantitative comparison of the resistance to Phytophthora root rot in three avocado rootstocks. Phytopathology 75:230-234.

Podger, F.D. 1972. Phytophthora cinnamomi, a cause of lethal disease in indigenous plant communities in Western Australia. Phytopathology 62:972-981.

Snyman, A.J., C.P. Snyman, and J.M Kotzg. 1984. Pathogenicity of avocado root rot fungi to Edranol seedlings and Duke 7 rooted cuttings. S. A. Avocado Growers' Assn. Yrbk. 7:80-81.

Weste, G., 1974. Phytophthora cinnamomi - The cause of severe disease in certain native communities in Victoria. Austral. Journal of Botany 23:67-76.

Zentmyer, G.A. 1984. Avocado Diseases. Tropical Pest Management 30:388-400.

Table 1. Lesion length on excised root tips of three avocado rootstocks inoculated with P. cinnamomi after 24 hours.
Rootstock	Lesion length after 24 h (mm)
	Inoculum type
	Mycelium suspension	Zoospore suspension
Edranol	5.8 a^z	2.6 abc
Duke 7	1.6 bc	1.8 bc
G755	0.0 c	0.0 c
^z Each value is the mean of five replicates. Values in horizontal and vertical columns not followed by the same letter differ significantly according to Duncan's multiple range test, (P<0.05).

Table 2. Linear colonization of excised root tips of three avocado rootstocks inoculated with P. cinnamomi after 24 hours.
Rootstock	Linear colonization after 24 h (mm)
	Inoculum type
	Mycelium suspension	Zoospore suspension
Edranol	4.0 bcd^z	4.8 bcd
Duke 7	7.2 abc	12.0 a
G755	1.6 cd	0.0 cd
^z Each value is the mean of five replicates. Values in horizontal and vertical columns not followed by the same letter differ significantly according to Duncan's multiple range test, (P<0.05).

Table 3. Lesion length on excised root tips of three avocado rootstocks inoculated with P. cinnamomi after 48 hours.
Rootstock	Lesion length after 48 h (mm)
	Inoculum type
	Mycelium suspension	Zoospore suspension
Edranol	15.6 ab^z	17.0 ab
Duke 7	13.4 b	12.0 b
G755	0.8 c	0.2 c
^z Each value is the mean of five replicates. Values in horizontal and vertical columns not followed by the same letter differ significantly according to Duncan's multiple range test, (P<0.05).

Table 4. Linear colonization of excised root tips of three avocado rootstocks inoculated with P. cinnamomi after 48 hours.
Rootstock	Linear colonization after 48 h (mm)
	Inoculum type
	Mycelium suspension	Zoospore suspension
Edranol	21.6^z	19.2 ab
Duke 7	21.6 a	30.4 a
G755	6.4 bc	0.0 c
^z Each value is the mean of five replicates. Values in horizontal and vertical columns not followed by the same letter differ significantly according to Duncan's multiple range test, (P<0.05).