Cucurbit Genetics Cooperative Report 18:26-28 (article 12) 1995
Qing Ma, Hongwen Cui
Department of Plant Protection, Northwestern Agricultural University, Yangling, Shaanxi 712100, P. R. China; Department of Horticulture, Northwestern Agricultural University, Yangling, Shaanxi 712100, P.R. China
Downy mildew, Pseudoperonospora cubensis (Berk. et Curt.) Rastow, is the most destructive cucumber disease in China. Breeding for disease resistance is the most effective control method. Although a number of resistant cultivars have been developed, mechanisms of resistance are not clear. Considerable experimentation has been directed towards the elucidation of Pseudoperonospora cubensis resistance in species other than cucumber (1, 3, 6). Riggle and Dunleavy (1981) studied the histology of leaf infection of susceptible and resistant soybeans by Peronospora manshurica (4). However, limited research has been carried out on downy mildew of cucumber in China. Li, et al. (1991) discussed changes in the host-pathogen relationship but failed to study the changes of the fungus itself (5). In order to provide a scientific basis for downy mildew resistance in China, we studied the resistance mechanism of various Chinese cucumber cultivars.
Materials and Methods.
Plants and pathogen. Four cucumber cultivars [Jinzha-2 (resistant), Jinyan-6 (moderately resistant), Heidan-1 (moderately susceptible), and ‘Changchun Mici’ (susceptible)] were grown in a greenhouse. An isolate of Pseudoperonospora cubensis obtained from infected cucumber plants in the field was maintained on cucumber plants growing in a growth chamber.
Inoculation. Before inoculation, spores on all leaves were removed by washing with clean water. Plants were then maintained at 100% RH for 24 h. The freshly produced sporangia were gently brushed off into distilled water. After a suspension of sporangia was sprayed on the 2nd leaf of plants at the 4-leaf stage, plants were held at 100% RH for 16 h and then transferred to chambers and grown under 10,000 lux irradiance for 16h at 18-22C for the duration of the experiment.
Sampling, staining and microscopic observation. Samples taken at 4, 6, 12, 24, 48, 72 and 96 h after inoculation were made transparent with saturated trichloracetaldehyde monohydrate, then stained with 0.1% lactophenol-cotton blue solution for 15 min. Penetration, mycelial growth, haustorium formation and plant cell necrosis were observed during light microscopy. Spore germination on leaf surfaces was observed after calcofluor staining under fluorescence microscopy.
Mycelial growth and haustorium development. After penetration, the fungus produced intercellular hyphae. Valvate or spherical haustoria were then produced from each intercellular hypha. Several haustoria formed in a single host cell. Occasionally substomatal vesicles were produced directly by inoculation. However, the average number of haustoria was distinctly higher on susceptible leaves than on resistant leaves. There were an average of 0.70 haustoria per penetration in susceptible leaves by 6 h after inoculation, whereas resistant leaves had only 0.21 haustoria per penetration. Similarly, by 48 h there were 11.17 and 2.53 haustoria observed per penetration on the leaves of susceptible and resistant cultivars, respectively (Table 1).
The intercellular hyphae penetration was slow until 24 h after inoculation, and was extremely rapid thereafter (48 h). Cultivars differed markedly in mycelial growth. At 48 h, the mycelium was 164.6 m long in susceptible leaves, and 64.7 m long in resistant leaves (Table 2). The mycelial growth on moderately-resistant and moderately-susceptible cultivars fell between the extreme values. Mycelial growth tended to accord with haustorium formation. With mycelial growth, more haustoria were formed.
The necrosis of leaf cells. During the progress of infection, rapid necrosis of host cells is an important event which relates to the expression of resistance. It was observed that leaf cell necrosis occurred in resistant and moderately resistant cultivars by 24 h after inoculation. In contrast, necrosis commenced in moderately susceptible and susceptible cultivars at 48 h and 72 h, respectively. By 72 h after inoculation, 13.7% of the penetration sites had necrosis cells in resistant leaves, with 40.7% in susceptible leaves. Data indicate that the time and rate of host cell necrosis differed between resistant and susceptible cultivars.
Discussion.
Cohen (1981) reported that Pseudoperonospora cubensis could infect the leaves both of susceptible and resistant cucumber cultivars, and produce intercellular hyphae and haustoria (2). The results of our experiment confirm Cohen’s observation, but are contrary to the argument of Li et al. (1991) which suggests that the infection process does not occur in resistant leaves (5). We have, however, found that the histopathological characteristics of leaves which were infected by P. cubensis differed among cucumber cultivars.
The formation of haustoria marks the establishment of parasitic relationship between the fungus and its host plant. Although every cultivar had formed haustoria by 6 h after inoculation, 65.6% of the penetration sites formed haustoria on susceptible leaves, only 21% was observed on resistant leaves. This results shows that, at the early stage of haustorium formation, a resistant cultivar has already expressed its resistance to P. cubensis. Compared with the infection on the susceptible cultivar, the process of infection is apparently inhibited on resistant cultivars. The formation of haustoria in leaves of resistant cultivars is slower and haustoria are less in number. Mycelial growth eventually slows down and fungal growth stops, at which time tiny spots appear on the plant’s surface. These histological characteristics only mirror processes involved in infection inhibition. The exact biological mechanism for such inhibition are not clear.
Li et al. (1991) observed cell necrosis in infected resistant cultivars. We found that host cell necrosis existed in the susceptible cultivar as well as the resistant cultivar, although the time and incidence of the necrosis differed distinctly among cultivars. Cell necrosis occurred at 24 h in the resistant cultivar, but at 72 h in the susceptible cultivar. In addition, the number of necrotic cells in the susceptible cultivar is very limited and did not affect the growth of the fungus. The earlier that necrosis occurs, the less the number of haustoria and the stronger the host resistance. Whether the necrosis of host cells is the cause/consequence of earlier inhibition or the death of the invading fungus and the histopathological characteristics of immune cultivars and non-host plant resistance is not clear.
Table 1. Comparison of haustoria of Pseudoperonospora cubensis among different cucumber (Cucumis sativus L.) cultivars.
Average number of haustoria per penetration* |
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Resistance level |
Cultivar |
6h |
12h |
24h |
48h |
72h |
96h |
| Resistant | Jinzha-2 | 0.21 | 0.66 | 0.91 | 2.53 | 3.82 | 4.68 |
| Moderately resistant | Jinyan-6 | 0.55 | 1.02 | 1.26 | 4.15 | 6.33 | 7.71 |
| Moderately susceptioble | Heidan-1 | 0.63 | 1.12 | 1.64 | 8.15 | 10.90 | 13.52 |
| Susceptible | Changchun Mici | 0.70 | 1.38 | 2.15 | 11.17 | 13.25 | 15.57 |
*Thirty penetration sites were investigated at each sampling time.
Table 2. Mycelial growth on various cucumber (Cucumis sativus L.) cultivars after infection with Pseudoperonospora cubensis.
Mycelial length at various times after inoculation ( μ m)* |
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Resistance level |
Cultivar |
24h |
48h |
72h |
96h |
| Resistant | Jinzha-2 | 16.4 | 64.7 | 73.9 | 82.3 |
| Moderately resistant | Jinuan-6 | 19.5 | 75.0 | 98.1 | 107.4 |
| Moderately susceptible | Heiden-1 | 25.4 | 117.6 | 127.8 | 176.2 |
| Susceptible | Changchun Mici | 27.9 | 164.4 | 176.2 | 190.7 |
*Thirty penetration sites were investigated at each sampling time.
Literature Cited
- Chou, C.K. 1970. An electron microscope study of host penetration and early stages of haustorium formation of Peronospora parasitica (Fr.) Tul. on cabbage cotyledons. Ann. Bot. 34:189-204.
- Cohen, Y. 1981. Downy mildew of cucurbits. In: D.M. Spencer (ed.), The Downy Mildews. Academic Press, London. pp. 341-354.
- Crute, I.R. and G.R. Dixon. 1981. Downy mildew diseases caused by the genus Bremia Regel. In: D.M. S[emcer (ed.), The Downy Mildews. Academic Press, London. pp. 515-529.
- Dunleavy, J.M. 1981. Downy mildew of soybean. In: D.M. Spencer (ed.), The Downy Mildews. Academic Press, London. pp. 515-529.
- Li, J. et al. 1991. Microscopial and ultrastructural studies on the resistance of cucumber to Pseudoperonospora cubensis. J. Wuhan Botn. Res. 9:209-214.
- Royle, D.J. and H.R. Krembeller. 1981. Downy mildew of the hop. In: D.M. Spemcer (ed.), The Downy Mildews. Academic Press, London. pp. 395-419.