African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 6, Num. 4, 1998, pp. 345-350

M.A. OKIROR

Department of Botany, Egerton University, P. O. Box 536, Njoro, Kenya

(Received 4 August, 1997; accepted 21 December, 1998)

Code Number:CS98036

ABSTRACT

A repeatable inoculation procedure for Fusarium wilt of pigeonpea caused by Fusarium udum Butler was developed. Five techniques, namely, sowing seeds or transplanting seedlings into infested soil, dipping roots or soaking seed in a spore suspension, and stem injection were tested under glasshouse conditions on four cultivars of pigeonpea with different levels of resistance. Sowing seed in infested soils gave the highest mortality and allowed for easy differentiation of resistant and susceptible plants. The other techniques either gave severe wilting, inconsistent results or low wilting, and were considered unreliable. This study recommends that sowing seed in infested soil in a glasshouse be adopted as a standard procedure for scoring this disease.

Key Words: Cajanus cajan, Fusarium udum, inoculation technique, root dipping, spore concentration, stem injection

Une procédure d'inoculation périodique du Fusarium wilt de Pigeonpea causé par le Fusarium udum Butler a été développée. Cinq techniques, notamment la semence de grains ou la transplantation des semis dans un sol infecté, le trempage des racines ou des grains dans une suspension de spores, et l'injection de la tige étaient testées dans des conditions de serre sur quatre variétés cultivées de pigeonpea aux différents niveaux de resistance. La semence de grains dans un sol infecté a donné une mortalité très élevée et a permis une différenciation aisée sur des plants résistants et susceptibles. Les autres techniques ont donné soit une sévère fanaison, soit des résultats inconsistants ou un léger dessèchement, et ont été considérées comme moins confiantes. Cette étude recommande que la semence des grains dans un sol infecté placé dans des conditions de serre soit adoptée comme une procédure modèle pour combattre la maladie.

Mots Clés: Cajanus cajan, Fusarium udum, techniques d'inoculation, trempage de racine, concentration de spore, trempage de tige xxxx

The success of an exercise to identify pigeonpea (Cajanus cajan (L.) Millsp.) germplasm resistant to Fusarium wilt caused by Fusarium udum Butler depends heavily on the procedure used. Pigeonpea has traditionally been screened for wilt resistance in wilt-infested fields (Butler, 1908; Deshpande et al., 1963). The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) also uses field screening but has reported cases of inconsistent results (Nene et al., 1981). They are also testing other techniques, especially for use in the glasshouse.

Various techniques have been used for glasshouse screening for resistance to soil-borne pathogens, including root dipping in inoculum (Phipps and Stipes, 1973), growing seed or seedlings in infested soils (Russell, 1978), soaking seed in inoculum (Sakar et al., 1982) and injecting inoculum into plants (Jindal et al., 1982). The effectiveness of these techniques depends on various factors, including spore concentration, plant age at inoculation, and environmental conditions such as temperature and humidity (Ribeiro and Hagedorn, 1979). Plant injury, especially to the roots, enhances infection (Henderson and Winstead, 1961). The objective of this study was to find a suitable glasshouse technique for screening pigeonpea germplasm for resistance to Fusarium wilt.

Cultivars. Four pigeonpea cultivars - NPP 679, ICP 2376, ICP 2376, ICP 270 and Munaa - were used in this study. Two of these, NPP 679 and Munaa, are local cultivars. The former is resistant to Fusarium wilt while the latter is susceptible. The other two, ICP 2376 and ICP 270 are from ICRISAT and are susceptible and resistant to wilt, respectively. All these cultivars had been selfed for four generations and were considered genetically homozygous.

Inoculum. Inoculum was prepared from wilted pigeonpea plants collected from Thika, a non-traditional pigeonpea growing area in the Kiambu district of central Kenya. Plant roots and stems were chopped and incorporated into large wooden boxes (94 x 50cm x 43cm deep) filled with forest soil in the glasshouse. One kg of plant material per 5 kg soil was put in the boxes. The boxes were watered lightly everyday, and more plant material was added at the rate of 1kg box-1 monthly until the boxes were confirmed to be infested with Fusarium. They were then designated sickboxes. Single spore cultures of F. udum were produced on potato dextrose agar (PDA), and used to prepare inoculum suspensions.

Inoculation techniques. Five techniques were compared, namely, (a) sowing seed in infested soil, (b) transplanting seedlings into infested soil, (c) soaking seed in inoculum, (d) dipping roots of seedlings in inoculum, and (e) stem injection of inoculum.

(a) Sowing seeds. Thirty seeds per cultivar were sown in a sickbox. Furrows were marked 7 cm apart across the box. Each cultivar was sown in three furrows (rows) at a rate of ten seeds per furrow. Non-infested soil in a similar box was used as the control.

(b) Transplanting into sick boxes. Twenty three-weeks old seedlings per cultivar grown in Fusarium-free soil were used. Roots were trimmed on ten seedlings but were left intact on the other ten. Seedlings were transplanted into furrows in the sickbox. Three furrows of seedlings were planted, each with intact and trimmed roots per cultivar. Seedlings treated similarly were planted in pathogen-free soil.

(c) Seed soaking. Four spore concentrations were used: 4.2 x 105, 9.4 x 105, 1.3 x 106 and 1.6 x 106 spores ml-1. Thirty seeds per cultivar were placed in a beaker containing about 200 ml of each of the inocula. At 1, 5 and 7 h after immersion, ten seeds were removed from each beaker and sown in a furrow in disease-free soil. Seeds soaked in water served as the control.

(d) Root dipping. Twenty three-weeks old seedlings of each cultivar were tested at each spore concentration used for seed soaking. Of these ten had their roots trimmed. Seedling roots were immersed in one of the four spore concentrations for 1.5 hr and transplanted into non infested soil in the boxes. Seedlings with roots dipped in water served as a control.

(e) Stem injection. Two spore concentrations were used: 1.3 x 106 and 1.6 x 106 spores ml-1, and control seedlings received water. 0.25 ml of inoculum in a hypodermic syringe was injected into a four-weeks old seedling, 4 cm above ground. Ten seedlings per cultivar were injected with each inoculum.

Methods (a) and (b) were repeated three times each while methods (c), (d) and (e) were repeated four times. All experiments were conducted in a glasshouse at the University of Nairobi, Kabete Campus. Day temperatures in the glasshouse were 38-42$C (maximum) and 25-29$C (minimum). Initial plant stands were recorded 14 days after either germination (if seed were sown), or when transplanted seedlings had survived (in case of transplants).

Data collection and analysis. The onset of wilting and the number of plants wilted was recorded weekly; final counts were taken three months after inoculations.

Characteristic wilt symptoms were observed where F. udum inoculum was used. No wilting appeared until at least four weeks after inoculation, and at least nine weeks after stem injection.

Direct sowing. The first wilted plant appeared four weeks after inoculation in Munaa, and was followed rapidly by more deaths in both Munaa and ICP 2376. Wilt was slow to occur and spread in ICP 270. NPP 679, originally considered resistant, broke down and was heavily wilted. The overall mean wilt caused by this technique was 56% and was the highest in all the methods studied. Susceptible and resistant cultivars were easily distinguished (Table 1).

Transplanting seedlings into sick boxes. Wilted plants first appeared in the fifth week in both Munaa and ICP 2376. By the end of week seven, the extent of wilting was much higher and all cultivars were affected, but the susceptible cultivars were more severely affected and distinguishable from the resistant cultivars. Again, NPP 679 was heavily affected. The average mortality was 46% for seedlings with intact roots and 51% for those with pruned roots; this difference was not statistically significant (Table 1). This technique was ranked third after direct sowing and root dipping.

Stem injection. No wilt was recorded until the ninth week when one plant of Munaa wilted. This plant had received the higher of the two inoculum concentrations. Eventually, a few wilted plants appeared in all of the cultivars. Affected plants were generally only partially wilted. This technique caused the least wilting (Table 1).

TABLE 1. Effect of inoculation method on Fusarium wilt incidence in four pigeonpea cultivars (% of plants wilted)

Root dipping. Like direct sowing, root dipping also led to onset of wilting four weeks after inoculation. The plants that wilted had their roots trimmed and dipped in the most concentrated inoculum. Wilting increased with time and in all cultivars and root treatments. By the end of the study, more wilted plants were recorded, especially at the highest inoculum level. At the three highest levels of inoculum separation of resistant and susceptible cultivars was difficult (Table 2). However, at the lowest inoculum concentration, resistant and susceptible cultivars reacted differently and distinctly although all mortalities were lower. Root injury did not give any major advantage over intact roots.

TABLE 2. Effect of root dipping seedlings on wilt incidence in four cultivars of pigeonpea (% of plants wilted)

Seed soaking. For all cultivars, wilting increased with increasing spore concentration and/or length of soaking. The mean % wilted plants for 1, 5, and 7 hr of soaking were 19, 19 and 26, respectively, and 13, 17, 23 and 28 for the four inoculum concentrations. This technique generally induced less wilting than the previous two methods (Table 3).

TABLE 3. Effect of soaking seed in different inoculum concentrations on wilt incidence in four cultivars of pigeonpea (% of plants wilted)

Several methods of inoculating pigeonpea with Fusarium were compared. Sowing seed directly into infested soil was the most effective. Wilting occurred early in the plant's growth, and the level of wilting induced was sufficient to differentiate between resistant and susceptible cultivars. Moreover, the technique is simple, reliable, easy to apply, and cost-effective and corresponds to sowing in infested soil in the field (Hubbeling, 1980). This technique has been reported to give the best comparison with field results in screening for resistance to Fusarium oxysporum f. vasinfectum in cotton (Hillocks, 1984). However, it is difficult to quantify the inoculum in the soil.

The root dip method resulted in a high percentage of wilted plants in all cultivars, especially at the three highest inoculum levels. However, it was considered more reliable at the low inoculum levels since the difference between susceptible and resistant cultivars was more apparent. Similar results with this technique have been reported by Phipps and Stipes (1973) for Mimosa. Root pruning was expected to predispose plants to early fungal invasion and lead to heavier wilting; however, the results showed no such a trend. This method was labour-and time-intensive and it may not be useful for screening large numbers of plants.

Wilting started late and progressed slowly in plants grown from seed soaked in various inoculum suspensions. The susceptible cultivars had 5 to 50% wilted plants while the resistant cultivars had 4 to 40%. This method, therefore, does not clearly separate resistant and susceptible pigeonpea plants.

Raising seedlings and then transplanting them into infested soil offered no signicant advantage over direct sowing of seed in such soil. It neither led to earlier occurrence of wilt nor induced higher levels of wilting even when seedlings had their roots pruned. Given that this technique requires equipment to grow seedlings, culture the pathogen, prepare and standardise the inoculum, it is not as useful as the direct sowing techique discribed above.

The results of stem injection suggest that it is not an effective method of screening as previously observed by Sharma et al. (1977). Besides inducing only very low wilting, it is slow and requires significant labour to inject the plants. Of the methods studied, stem inoculation was the least effective.

In conclusion, sowing seed into infested soil is the most effective approach for screening pigeonpea for resistance against F. udum. This technique is easy to carry out, gives dependable results and is similar to field screening except for controlled conditions. Root dipping and transplanting seedlings into infested soil, are also effective but have logistical shortcomings that make direct sowing of seed a most appropriate technique. Both seed soaking and stem injection were unreliable. For the immediate needs of screening pigeonpea germplasm for resistance to Fusarium wilt, glasshouse screening shall be adopted, based on the sickbox test.

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