Effect of root damage on the development of Fusarium wilt in peas

[ 577 ] Trans. Brit. mycol. Soc. 46 (4),577-584 (1963).

EFFECT OF ROOT DAMAGE ON THE DEVELOPMENT OF FUSARIUM WILT IN PEAS By D. A. DOLING

National Institute

of Agricultural Botany,

(With

2

Cambridge

Text-figures)

Fusarium oxysporum f. sp, pisi (Lindf.) Snyd. & Hans. caused wilt in peas only when the roots were mechanically damaged. The rate of wilting depended upon the severity of treatment used in damaging the roots. Isolation of the fungus from inoculated plants suggested that invasion occurred at the cut surface of the root.

In studies on pea wilt caused by Fusarium oxysporum f. sp. pisi most inoculation techniques have involved root damage, either intentionally or inadvertently. For example, Linford (1931) transferred germinating seeds and young seedlings to infested soil or cut the roots in situ before pouring the fungus suspension over the surface; Wells, Hare & Walker ( I 949) transplanted seedlings after either dipping in a spore suspension or cutting the roots that were submerged in a fungus suspension. Infection has also been obtained when seed was sown in soil infested with inoculated Melilotus stems (Linford, 1928) or with a liquid culture of the fungus (Wade, 1929) or with cornmeal/sand inoculum (Buxton, 1955). In preliminary experiments at the N.I.A.B. no wilt occurred when peas were sown in infested compost. Because of this, experiments were designed to examine the effects of root damage on wilt development. MATERIALS AND METHODS

All experiments were conducted in a heated glasshouse during AprilMay and August-October. William Massey (syn. Kelvedon Wonder) peas were grown from seed in boxes containing John Innes potting compost in which the loam had been previously steam sterilized. When the seedlings were about 2 in. high, 12-14 days after sowing, they were washed free from adhering compost, dipped in a suspension of F. oxysporum f. sp. pisi and transplanted into 5 in. pots, twelve plants in three pots comprising each treatment. After transplanting, 25 ml. of fungus suspension were poured about the plants in each pot. The suspension was prepared by culturing in Czapek-Dox liquid medium for 12 days at 26° C. and then filtering through coarse muslin. Disease assessments were made by determining the mean percentage of wilted leaves per plant. When using such a method of inoculation it is essential to know the effect of the culture medium on the development of wilt. This was ascertained by centrifuging part of the fungus suspension at 3500 r.p.m. for 10 min., decanting the supernatant liquid and resuspending in an 37

11yc. 46

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Transactions British Mycological Society

equivalent volume of distilled water. Wilt symptoms developed several days earlier in the plants inoculated with the fungus in the culture medium, but 27 days after inoculation the plants in the two treatments were equally wilted. The rate of wilt in seedlings transplanted into steam-sterilized and non-sterilized compost was also compared but no differences were detected.

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Effect of spore concentration on the development of wilt, height of plants and number of leaves.

The effect of spore concentration was also examined using a series of ten spore suspensions in which the concentration of each was half that of the previous one. Wilt was assessed 23, 31 and 62 days after inoculation and the number of leaves and height of the plants was also recorded (Fig. I). High concentrations caused wilt to develop more quickly and considerably dwarfed the plants by reducing the length rather than the number of internodes. In view of these results and the similar results of Buxton & Perry (1959) the standard procedure adopted was to use undiluted fungus suspension in the culture medium and steam sterilized soil in the compost.

Pea wilt. D. A. Doling

579

RATES OF WILT DEVELOPMENT

Comparison between transplanting and direct sowing Twelve seedlings were inoculated by the standard procedure and compared with a second series in which the fungus suspension was poured round the seeds at sowing. Wilt symptoms first appeared in the transplanted seedlings 20 days after inoculation, the percentage of leaves wilted rising to 64 % after 28 days and to 77 % after 34 days. The seedlings inoculated at sowing time and left in situ undisturbed did not wilt. Nine other experiments in which these two treatments were included confirmed these results. The absence of wilt in the directly sown seedlings could be caused by leaching of the fungus suspension or by its inactivation by the developing seedlings. To ascertain whether or not the fungus was present and able to cause wilt, three sets of pots were treated as follows: (a) 25 ml. fungus suspension was poured into four holes in each pot, the holes were filled in and marked and the pots watered normally. After 14 days seedlings were transplanted into the pots at each inoculum site. (b) The fungus suspension was added with the seed at the time of sowing. After 14 days the seedlings were removed and replaced by other 14-day-old seedlings which had been grown in clean compost. (c) Fungus suspension was added with the seed and 14 days later the seedlings were removed and replaced in their original position in the pots. Wilting occurred at almost the same rate after transplanting in each set (T able I), although the seedlings in method (c) had been in contact with the fungus 14 days longer than those in (a) and (b). Half the leaves wilted 20 days after transplanting and about three-quarters wilted after 26 days. Lack of wilt in the undisturbed seedlings could not, therefore, be attributed to leaching of the fungus suspension. Table

I.

Percentage of wilted leaves in transplanted and non-transplanted pea seedlings in sterilized compost Days after transplanting ~

Treatment Control

a b

Seed + inoculum Inoculum only; seedlings transplanted 14 days later Seed+inoculum; 14 days later , seedlings removed and replaced by others Seed+inoculum ; 14 days later , seedlings removed and replac ed

Effect

20

26

o 54

o 81

51

ofseuerity of root damage at transplanting

The previous experiments showed that wilt development is associated with transplanting. Damage to the roots was thought to be the causal factor so comparisons of wilt development in plants with severely, as opposed to slightly damaged, roots were made. Eight- and 14-day-old seedlings were transplanted into infested soil (a) with great care in order 37- 2

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Transactions British Mycological Society

to minimize root damage, and (b) after damaging all roots visible on the outside of the ball of soil by pinching the roots between the finger and thumb. Wilt occurred in all treatments (Table 2) but was significantly less (P = 0'05) at both assessments in the seedlings transplanted carefully when 8 days old. In the seedlings transplanted when 14 days old the differences were less marked, being near significance (P = 0'05) when assessed 26 days after inoculation and not significant (P = 0'05) after 35 and 49 days. Tests for significance were based on analysis after transformation to angular measure. Such results might be expected, as least damage would occur in the youngest seedlings carefully handled, whereas 14-day-old seedlings have a large root system and even with great care many roots would be inadvertently damaged. Table

2.

Percentage ofwilted leaves in seedlings transplanted with and without deliberate root damage Days after transplanting A

Method of transplanting 8-day old seedlings (a) Carefully (h) With damage 14-day old seedlings (a) Carefully (h) With damage

26

32

35

49

67

45 63 38 53

41

100

63

90 90

72

Table 3. Percentage wilted leaves 35 doss after inoculation following different root pruning treatments Inoculation treatment a

b

d

f

Standard procedure; 14-day-old seedlings washed in water and dipped in fungus suspension As (a) but all roots trimmed to I in. long before dipping and severed root pieces removed As (h) but severed root pieces added with the seedlings As (h) but roots cut when submerged below the surface of the fungus suspension As (a) but roots trimmed to 2-3 in. in length and severed root pieces removed As (e) but severed root pieces added with the seedlings

Percentage wilt 74 42 80 100

27 57

The effect of root damage was further investigated in a second experiment in which six treatments varying in the severity of root damage were used. The treatments and the percentage wilt occurring 35 days after inoculation are set out in Table 3. Wilt developed significantly more quickly (P = 0'05) than in any other treatment when the roots were cut whilst submerged in the fungus suspension (method (d)). Severe root pruning caused wilt more quickly (cf. method (b) with method (e) and method (c) with method (1)), the difference between (c) and (f) being significant (P = 0'05). The addition of the severed root pieces significantly

58r

Pea wilt. D. A. Doling

increased the rate of wilt development (cf. method (b) with (c), P = 0'05, and (e) with (1), P = 0'01). Tests for significance were based on analysis after transformation to angular measure.

Effect

ofdamaging roots in situ

When seedlings are transplanted, the rate of development of wilt is associated with the severity of root damage. Even when considerable care in transplanting is taken, it is inevitable that extensive root damage must occur. To see whether very slight root damage is sufficient to allow entry of F. oxysporum f. sp. pisi and to ensure that roots were undamaged in the control pots, roots of plants were damaged in situ. The fungus suspension was added with the seeds when sown and after 14 days roots were damaged in different ways: (I) a scalpel was plunged once, (2) a needle (0·6 mm. diam.) was plunged 2, 4 and 8 times, and (3) a needle (2 mm. diam.) was plunged 2, 4 and 8 times around each plant. Each insertion was made I in. from the stem down to the bottom of the pot. The percentage wilt was not significantly different in any of the treatments involving root damage. This demonstrates that the extent of the damage of individual plants over this range did not affect the rate of wilt development, but the number of plants showing wilt varied (Table 4). By contrast, in experiments where they were transplanted, all seedlings wilted and a high degree of uniformity was obtained. Table 4. Percentage wilted leaves 26 days after damaging roots in situ No. of wounding treatments per plant

,

A

Treatment

Undamaged Scalpel 0·6 mm. needle 2'0 mrn. needle

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I = no. of plants infected. treatment).

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58 63

8 9

71 52

percentage wilted leaves of infected plants (15 plants/

INVASION OF PEA ROOTS BY FUSARIUM OXYSPORUM F. SP. PISI

The association between wilting and root damage has been demonstrated but the experiments did not indicate where infection occurred. To ascertain the points of entry on the root by the fungus, seedlings were severely root pruned by cutting all roots to approx. I in. long before dipping in the fungus suspension, and then transplanted. At 3-day intervals five plants were removed from the pots, washed free from soil and surface sterilized for I! min. in I % AgNO a followed by two washes in saturated NaCI and two washes in sterile water. A series of 2 mm. segments of the tap root and four secondary roots were then plated on potato dextrose agar in order from the cut root end, and incubated for 6 days at 26° C. The number of infected segments was greatest near the

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Transactions British Mycological Society

damaged root end and, at a constant distance from the end, increased with the length of time the plants remained in the compost (Fig. 2). Although a few anomalous infections occurred (5th, 6th, 9th and loth segments were infected after 3 days), possibly owing to inadvertent root damage, these results show a trend suggesting that infection occurs through the cut surface at the end of the root rather than at random over its surface. 9 days

3 days

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2.

Frequency of isolation of F. oxysporum f. sp. pisi from pea-root segments 3, 9,

15

and

21

days after inoculation.

DISCUSSION

These experiments show that for wilt-susceptible William Massey pea seedlings grown in steam-sterilized compost (I) intact roots are not infected by F. o. f. sp. pisi; (2) mechanical damage is a pre-requisite for the development of wilt; (3) the severity of root damage influences the rate of wilting; and (4) the damaged root ends provide points of entry for the fungus. Several workers have studied the effect of root damage on the infection of roots by different forms of F. osysporum. Wade (1929) and Linford (193 I) found that no breakdown in resistance of resistant pea varieties resulted from damaging roots; the former reported that damaging in situ roots of peas of a susceptible variety in naturally infested soil did not cause earlier wilting, but in a comparison between susceptible seedlings transplanted with care and those given severe root pruning, wilting appeared distinctly earlier in the former. He concluded that wounding of roots was unfavourable to the development of the disease. However, the comparison he made was not between damaged and undamaged roots but rather between damaged roots with the prunings removed and damaged roots with dying root pieces still present, i.e. between treatments similar to (a) and (b) in Table 3. A similar experiment by Keyworth & Dimond (1952) showed that severe root pruning reduced wilting in tomato. The

Pea wilt. D. A. Doling inference from these experiments is, therefore, that different root-damaging treatments cause different rates of wilting in susceptible varieties. Wardlaw (193 I) and Sequeira, Steeves, Steeves & Riedhart (1958) found that F. o. f. sp. cubense did not invade actively growing banana roots, though Sequeira et al. secured infection when mature root tissues were damaged so as to expose the vascular tissues but not when immature tissues were damaged. This observation is consistent with that of Riopel (1960) who noted that wound reaction of banana roots was vigorous only in the immature portions of the stele. If similar wound reaction differences occur in pea roots the reduction of wilt in the trimmed root treatment as compared with the severe root pruning (Table 3) could be explained. Invasion of fungi following damage by plasmolysing the root tissues has also been studied. Rishbeth (1955) obtained a reduction in the number of banana root infections after flooding with a salt solution or using hot water. Reduced wilting in tomatoes was also reported by Keyworth & Dimond (1952) after treatment with hot water or a number of chemical compounds. It appears that these two methods of root damage, by mechanical means and by plasmolysis, can influence wilting in opposite ways. The exposure of the stele to direct invasion by fungi presents an entirely different situation from that in which the root, when killed by plasmolysis, has the stele enclosed within a mass of dead cell tissues. The resistance mechanisms operative in the outer tissues of the healthy intact root may still be active in the plasmolysed tissues. Alternatively, competitive activity on the part of the many soil organisms in these cells could prevent access of the pathogen to the stele. When the stele is exposed entry of the fungus is very rapid (Fig. 2); it could be isolated 3 days after inoculation. In view of this, the role of the severed root pieces left in situ is not clear, for it is unlikely that they could selectively aid the pathogen in the short time available before invasion of the stele occurs. Although this series of experiments presents evidence for the rapid invasion of the exposed stele, the possibility of invasion at other points should be considered, since Linford (1928), Wade (1929) and Buxton (1955) obtained wilt without causing any root disturbance. Infection of the root tip and region of differentiation with F. o. f. sp. callistephi by Ullstrup (1937) with F. o. f. sp. conglutinans by Peterson (1960), and with F. o. f. sp. cubense by Stover (1962) has been described. Virgin & Walker (1940) reported penetration by F. o. f. sp. pisi most commonly through the root tip and cotyledonary node of peas grown on agar. Hepple (1960), using naturally infested soil in a glasshouse, concluded that invasion by F. o. f. sp. pisi is mainly through moribund cotyledons and later obtained evidence to show that the growth of the fungus on pea cotyledons is greatly stimulated by the presence of bacteria and nematodes (Hepple, 1963). Most of the available evidence on the effect of root damage on vascular wilts is based on experiments involving artificial inoculation, and it is a matter of conjecture as to how invasion occurs under natural conditions. It would appear that an exposed stele is necessary for the entry of the pathogen but the means by which such a condition is effected in the soil is not known.

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The writer wishes to express his thanks to Mr M. H. Meadway for assistance with the routine work. REFERENCES

BUXTON, E. W. (1955). Fusarium diseases of peas. Trans. Brit. mycol. Soc. 38, 309-316. BUXTON, E. W. & PERRY, D. A. (1959). Pathogenic interactions between Fusarium oxysporum and F. solani on peas. Trans. Brit. mycol. Soc. 42, 378-387. KEYWORTH, W. G. & DIMOND, A. E. (1952). Root injury as a factor in the assessment of chemotherapeutants. Phytopathology, 42, 311-315. HEPPLE, S. (1960). Infection of peas by wilt disease fungi. Nature, Lond., 185, 333-334. HEPPLE, S. (1963). Infection of pea plants by Fusarium oxysporum f. sp. pisi in naturally infested soil. Trans. Brit. mycol. Soc. 46, 585-594. LINFORD, M. B. (1928). A Fusarium wilt of peas in Wisconsin. Res. Bull. Wis. agric. Exp. Sta. 85, 44 pp. LINFORD, M. B. (1931). Studies of pathogenesis and resistance in pea wilt caused by Fusarium orthoceras f. pisi. Phytopathology, 21, 797-826. PETERSON, J. L. (1960). Studies on resistance in radish to Fusarium oxysporum f. conglutinans race 2. Diss. Abstr. 20, 2483-4. (Seen in Rev. appl. Mycol. 39, 753.) RIOPEL,J. L. (1960). Studies on development and wound responses of the roots of Musa 'Gros Michel' in relation to the Panama disease. Ph.D. Thesis, Harvard Univ. (Seen in Phytopath. Pap., C.M.I. 4, 1962.) RISHBETH, J. (1955)' Fusarium wilt of bananas in Jamaica. I. Some observations on the epidemiology of the disease. Ann. Bot., Lond. N.S. 19, 293-328. SEQUEIRA, L., STEEVES, T. A., STEEVES, M. W. & RIEDHART, J. M. (1958). Role of root injury in Panama disease infections. Nature, Lond., 182, 309-31 r. STOVER, R. H. (1962). Fusarial wilt (Panama disease) of bananas and other Musa species. Phytopath. Pap. C.M.I. 4, 1-32. ULLSTRUP, A. J. (1937). Histological studies on wilt of China aster. Phytopathology, 27, 737-748. VIRGIN, W.J. & WALKER,]. C. (1940). Relation of the near wilt fungus to the pea plant. J. agric. Res. 60, 241-248. WADE, B. L. (1929). Inheritance of Fusarium wilt resistance in canning peas. Res. Bull. Wis. agric. Exp. Sta. no. 97, 32 pp. WARDLAW, C. W. (1931). The biology of banana wilt (Panama disease). III. An examination of sucker infection through root bases. Ann. Bot., Lond. 45, 383-399. WELLS, D. G., HARE, W. W. & WALKER, J. C. (1949). Evaluation of resistance and susceptibility in garden pea to near wilt in the greenhouse. Phytopathology, 39,

771-779.

(Accepted for publication

21

March 1963)