Partial resistance to a Fusarium root disease in Egyptian white lupin landraces

Euphytica 112: 233–237, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

233

Partial resistance to a Fusarium root disease in Egyptian white lupin landraces S. Raza, J.L. Christiansen, B. Jørnsgård & R. Ortiz Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK 1871 Frederiksberg C, Denmark Received 8 September 1998; accepted 19 September 1999

Key words: Egypt, Fusarium sp., landraces, Lupinus albus, resistance breeding

Summary A Fusarium sp. root pathogen of lupin is the causal agent of the most important disease that affects the cultivation of white lupin (Lupinus albus L.) in Egypt. The aim of our research was to investigate whether host resistance to Fusarium root disease was available in Egyptian landraces of Lupinus albus. Five Fusarium isolates collected from white lupin samples in Egypt were tested with an Egyptian landrace and a French cultivar. The most aggressive isolate was used to screen an additional 15 Egyptian landraces, two cultivars released in Egypt after selection among landraces, one Polish cultivar, and two French cultivars. The assessment of host response to Fusarium was performed in a field, and under controlled conditions in a greenhouse pot experiment. Most landraces and the two Egyptian cultivars showed better resistance with good accordance between field and pot experiment. This experiment showed that Egyptian genetic resources of white lupin possess partial resistance to Fusarium root rot. Egyptian germplasm may be an alternative genetic source for incorporating partial resistance to Fusarium root rot into the breeding pools.

Introduction Fusarium root rot is a major disease to white lupin in Egypt causing yield losses of up to 40% (Osman et al., 1986). Cultural practices, though important, are not always able to protect the crop against the disease (Fahim et al., 1986a; Fahim et al., 1986b; Osman et al., 1986). Hence, resistant cultivars provide the best control method for this disease. Resistance to Fusarium wilt in L. albus has been reported in Russian germplasm (Kurlovich et al., 1995). A few Ukrainian cultivars, such as ‘Druzba’, ‘Pischchevoy’, ‘Olezhka’, ‘Siniy Parus’, and ‘Dieta’ possess some resistance to this wilt disease (N. Kuporneychuk, Chabany, Kiev, unp. results). Some resistance was also observed in three white lupin landraces collected in Egypt (Raza et al., 1998). We have collected more Egyptian landraces, whose potential for plant breeding were in situ assessed recently (Christiansen et al., 1999). This paper reports the level of resistance, as determined in pot and field

experiments, of Egyptian landraces and cultivars to one of the most aggressive strains of Fusarium root rot pathogen isolated in Egypt.

Material and methods Samples of wilted white lupin plants were collected from farmer fields in different Egyptian locations. Roots and partial basal stems of diseased plants with typical symptoms of root-rot wilt and vascular discolourations were washed carefully with running tap water. Small plant pieces were sterilized in 2% sodium hyperchlorite for 3 minutes, washed twice in sterilized distilled water, and dried between two sterilized filter papers. Five sterilized samples were placed directly on petri dishes with potato dextrose agar (PDA) medium and incubated for 7 days at 25◦ C. The emergent fungi were isolated and purified on PDA plates for single spores, and subsequently identified according to their morphological characteristics with the aid of a mi-

234 Table 1. Analysis of variance of pathogenity test in clay soil and sandy soil Source

Replication Accession Isolate Accession∗ isolate Error

d.f.

3 1 5 5 32

Clay soil Early wilt m.s.

Late wilt m.s.

Sandy soil Early wilt m.s.

Late wilt m.s.

36 ∗ 3 n.s. 2537 ∗∗∗ 144 ∗∗∗ 7

9 n.s. 453 ∗∗∗ 3066 ∗∗∗ 233 ∗∗∗ 8

16 n.s. 1255 ∗∗∗ 4646 ∗∗∗ 382 ∗∗∗ 37

87 n.s. 231 ∗ 5599 ∗∗∗ 111 n.s. 52

croscope and following available descriptions (Smith, 1965; Booth, 1971; Nelson et al., 1983). Stock cultures were stored in a plant pathology herbarium for future studies. Pathogenecity tests were carried out to determine the pathogenic potential of five distinct Egyptian isolates of Fusarium f. sp. Lupini in clay pots of 25 cm diameters. These pots were filled with sterilized clay or sandy soils, and infested with each isolate using the spore suspension technique of Wensley & Mckeen (1962). Conidial suspensions were obtained after flooding 12 day-old Fusarium sp. cultures with sterile water and filtering through three layers of cheese-cloth. The concentration of conidial suspension was adjusted according to counts with hemacytometer. Each pot was infested with 20 ml of conidial containing 1 × 106 spores per millilitre. A French cultivar (‘Alex’) and an Egyptian landrace (accession 8) were grown in these infested pots in a greenhouse at variable temperature (20–30◦C), relative humidity (40–80%) and fertilized once a week with 50 ml of a 20–20–20 (NPK, 3 g/l) fertilizer suspension. Non-infested pots of each accession were added as controls. The experiment had four replications with 10 seeds in each pot. The experiment was sown on 10 December 1996. The percentage of early and late wilt were recorded 4 and 12 weeks after infestation. Screening for resistant accessions was carried out in the greenhouse and in the field. Twenty accessions including 15 landraces and two cultivars released in Egypt (Giza1 and Giza2), one Polish cultivar (‘Wat’), and two French cultivars (‘Alex’ and ‘Athos’) were tested for their response to the most aggressive Fusarium isolate. Percentage of early and late wilt were counted in each pot following the above experimental protocol. Disease severity was further estimated on remaining plants using an arbitrary scale of 0 (no visible symptoms), 1 (wilting symptoms just visible), 2 (clear wilting symptoms on several plants),

Table 2. Percentage of early and late wilt in a resistant and susceptible white lupin accession tested against five Egyptian isolates of Fusarium sp. Accession

Isolate Clay soil Sandy soil Early wilt Late wilt Early wilt Late wilt % % % %

Landrace 8z 1 2 3 4 5 Control ‘Alex’ 1 2 3 4 5 Control

5.8 10.5 17.5 9.8 7.7 0.0 4.2 7.5 15.3 13.5 10.7 0.0 0.5

10.2 12.3 20.5 12.3 11.5 2.5 5.0 6.7 18.3 15.2 12.7 0.0 0.5

12.5 14.7 25.2 20.5 21.6 1.0 13.7 15.2 20.7 15.2 11.2 0.0 1.1

13.3 12.8 25.6 21.2 22.7 1.7 14.0 15.0 22.0 20.2 19.0 0.0 1.3

z Collection number in the Egyptian gene bank.

3 (half of the plants showed sever wilting), 4 (nearly all plants showing sever wilting), 5 (all plants wilting). Clay soil was used in the pot experiment in greenhouse, as the dominant soil type in the agricultural area in Egypt is clay. The sowing date was 20 November 1997, otherwise the experimental conditions were similar to the conditions in the pathogenecity test. In the field experiment the same twenty accessions were planted in a loamy-soil at Giza governorate naturally infested with Fusarium species. In addition, the field plots were infested with the Egyptian isolate 3 by mixing it with the soil. The experiment lay in a completely randomized design with three replications. Each plot was composed of two rows of 3 m with a row spacing of 0.6 m (i.e., plot size 6.2 m2 ). Seeds were sown in hills 15 cm apart with 2 seeds per hill,

235 Table 3. Analysis of variance of pot and field screening for fusarium resistence Source

Accession Replication Error

Pot experiment Early wilt Late wilt

d.f. 19 3 57

m.s. 73 n.s. 287 ∗ 73

m.s. 792 ∗ 3298 ∗∗∗ 382

Visualscore m.s. 2.298 ∗∗∗ 0.615 n.s. 0.690

d.f. 19 2 38

Field Early wilt

Late wilt

Visualscore

m.s. 443 n.s. 1348 ∗ 256

m.s. 780 ∗∗∗ 314 n.s. 205

m.s. 1.82 ∗∗∗ 1.08 n.s. 0.63

on 21 November 1997. Basal doses of N, P and K were added to all experimental plots at the rate of 30 kg of N (as urea 46% N), 60 kg of P (15.5% P2 O5 ) and 40 kg of K (48% K2 O) respectively. The experiments were irrigated twice. First irrigation took place immediately after sowing and the second just before flowering. The GLM procedure was used in the statistical analysis (SAS Institute, 1988).

Results and discussion The pathogenecity test revealed significant differences in the host response of white lupin accessions to late wilt of five isolates of Fusarium sp. on both soil types (Tables 1 and 2). Both the Egyptian landrace 8 and ‘Alex’ were most susceptible to isolate 3 on clay soil and to isolates 3, 4 and 5 on sandy soil. Therefore, isolate 3 was regarded the significantly most aggressive isolate in both clay and sandy soils, and it was chosen for further screening of other white lupin accessions. In the pot experiment there were significant differences among accessions for percentage of late wilting and the visual disease score using isolate 3 (Tables 3 and 4). All accessions had statistically similar percentages of early wilting (0–15%), suggesting that early screening does not provide good assessment of host response to this aggressive Egyptian isolate 3 of Fusarium sp. Most of the Egyptian landraces showed partial resistance to Fusarium sp. Highest resistance in the pot experiment was found in Giza 1 and Giza 2 (Table 4). The most susceptible variety was the French cultivar ‘Athos’. Results thus indicate that ‘Athos’ may be a more suitable, susceptible check variety than ‘Alex’. In the field experiment, the accessions were significantly different for their percentage of late wilt as well as in the visual disease scores (Tables 3 and 4). The late wilt score was generally higher in the field

Figure 1. Late wilt in field experiment plotted versus late wilt in pot experiment; values are means over replicates. ( Egyptian accession, # French and Polish accessions).

experiment than in the pot experiment, but a good correlation was found between the two experiments for the Egyptian accessions and varieties, with the exception of Acc32, which was less affected in the field than in the pot experiment. In a similar way also the 3 exotic varieties ‘Athos’, ‘Alex’ and ‘Wat’ deviated from the observed relation between field and pot late wilt severity by having a relatively lower field infection (Figure 1, Table 4). Both in field and pot experiment ‘Athos’ was recorded the most susceptible variety, and Giza 1 and Giza 2 among the most resistant. The Egyptian cultivars Giza-1 and Giza-2 were developed about 30 years ago in the Agriculture Research Center in Egypt using local landrace germ plasm. For future work, we recommend to do the preliminary screening in the greenhouse, and fur-

236 Table 4. Percentage of early wilt, late wilt and disease severity to Egyption isolate 3 of Fusarium sp. in the pot and field screening. Disease severity is recorded in a 0 (no visible syptoms) to 5 (dead or severely infected plant) scale Accession

Pot experiment Early wilt Late wilt (%) (%)

Disease score

Field experiment Early wilt Late wilt (%) (%)

Disease score

Landrace 4z Landrace 7 Landrace 11 Landrace 13 Landrace 15 Landrace 20 Landrace 24 Landrace 26 Landrace 28 Landrace 30 Landrace 32 Landrace 40 Landrace 41 Landrace 43 Landrace 44 Wat Alex Athos 2 Giza 1 Giza 2 Mean

5 5 10 5 0 10 5 0 0 5 0 10 5 5 5 0 15 10 10 5 5.5

25 35 50 30 50 35 40 40 45 45 55 30 30 25 45 55 40 75 15 20 39.3

2.05 1.20 1.15 1.65 1.75 1.45 1.45 1.25 1.75 1.65 1.55 1.65 3.70 3.25 2.60 2.85 2.00 1.50 0.85 0.85 1.81

8.3 0.0 33.3 12.5 25.0 12.5 16.7 16.7 33.3 12.5 20.8 12.5 16.7 45.8 29.1 29.2 41.7 25.0 12.5 4.2 20.4

45.8 70.8 70.8 62.5 58.3 58.3 66.7 58.5 54.2 54.2 37.5 54.2 50.0 33.3 66.6 20.8 25.0 75.0 29.2 37.5 51.5

1.4 2.1 3.3 3.2 2.9 2.0 1.7 2.1 2.7 1.5 3.6 2.8 2.7 2.4 0.5 1.1 0.8 – 2.4 2.0 2.2

LSD

N.S.

27.7

1.18

26.5

23.7

1.4

In the pot experiment uninoculated control plants of all accessions remained free of Fusarium sp. z Collection number in the Egyptian gene bank.

ther testing in the field to identify resistant genotypes to the local isolates of the pathogen. Following this procedure, many accessions may be tested by using the cheap greenhouse screening and only a few, which may have partial resistance as determined by the pot experiment, will be screened in the more expensive field testing. The more severe disease attack in the field experiment may partly be ascribed to different environmental conditions and partly to uncontrolled pathogens in the soil, including different races of Fusarium. Lamberts (1955) found that the severety of fusarium and the ratio of susceptible to resistant changed with temperature in a segregating population. It has earlier been shown that F. oxysporum sp. is the main agent causing wilt in lupin in Egypt, other isolated fungi, such as Cephalosporium maydis, Fusarium solani and Rhizoctonia solani only cause damping-off and root rot in lupin (Fahim et al., 1986b). The non-significant effect

of early scoring in the present experiment indicates that the main effect also in the field experiment can be ascribed to F. oxysporum. In Eastern Europe several species of Fusarium are reported to cause root rot in lupin (Debelyi et al., 1977), and several physiological races of F. oxysporum f.sp. lupni have been identified (Fugal Wegrzycka, 1984). Further work will aim at identifying the causal pathogens in Egypt. Single seed descent plants have been selected from these resistant accessions for further investigation on the genetics of host plant resistance to Fusarium root disease in this Egyptian white lupin germplasm. Likewise, this germplasm may provide the source population for enhancing, through cross breeding, the current level of partial resistance against this root rot disease.

237 Ackowledgements Germplasm collection of the Egyptian accessions was carried out as part of a joint research project between the Agricultural Research Center (ARC), Egypt and the Royal Veterinary and Agricultural University (KVL), Denmark with financial support from Danida.

References Booth, C., 1971. The genus Fusarium. Comm. Mycology Inst., Kew, Surrey, UK. Christiansen, J.L., S. Raza & R. Ortiz, 1999. White lupin germplasm collection and preliminary in situ diversity assessment in Egypt. Genetic Resources and Crop Evolution 46: 169–174. Debelyi, G.A., A.V. Zekunov & A.K. Minenko, 1977. Lupin mutants resistant to Fusarium diseases. Selectsiya i Semenovodstov 6: 63–64 (in Russian). Fahim, M.M., A.R. Osman, A.F. Sahab & M.M.A. El-Kader, 1986a. Agricultural practices and fungicide treatments for the control of Fusarium wilt of lupin. Egypt J Phytopathol 15: 45–46. Fahim, M.M., A.F. Sahab, A.R. Osman & M.M.A. El-Kader, 1986b. Studies on some soil-borne fungi attacking lupin plant. Egypt J Phytopathol 15: 17–25.

Furgal Wegrzycka, H., 1984. Occurrence and variation of the fungus Fusarium oxysporum Schl. f.sp. lupini Snyd. et Hans. in lupin in the Olsztyn region in the years 1972–1976. Roczniki Nauk Rolniczych, E Ochrona Roslin 14: 163–174 (in Polish). Kurlovich, B., N.S. Korneichuk & I.I. Kiselev, 1995. Breeding Fusarium resistant lupin forms for agricultural environment of Russia based on ecologico-geographical approach. J Appl Genet 36: 241–249 (in Russian). Lamberts, H., 1955. Broadening the bases for the breeding of yellow sweet lupine. Euphytica 4: 97–106. Nelson, S.E., J.A. Toussoun & W.F. Marasas, 1983. Fusarium spp.: an illustrated manual for identification. The Pennsylvania State Univ. Press, University Park, USA. Osman, A.R., M.M. Fahim, A.F. Sahab & M.M.A. El-Kader, 1986. Losses due to wilt of lupin. Egypt J Phytopathol 15: 27–34. Raza, S., A. Koriem, N. Abou-Zeid, J.L. Christiansen & B. Jørnsgård, 1998. Screening for resistance to wilt disease caused by Fusarium oxysporum in white lupin (Lupinus albus) in Egypt. Egypt J Appl Sci 13: 45–53. SAS Institute, SAS/STAT User’s Guide, Release 6.03 Edition, SAS Institute INC., Cary, N.C., 1988. Smith, H.C., 1965. The morphology of Verticillium albo-atrum, V. dahliae and V. tricorpuso. NZ Agric Res 8: 450–478. Wensley, R.N. & C.D. Mckeen, 1962. Rapid test for pathogenicity of soil isolates of Fusarium oxysporum f.sp. melonis. Can J Micr 8: 818–819.