Ann. appl. Biol. (1968), 61, 503-510

Printed in Great Britain 503

The interrelationships of the causal fungus of brown root rot of tomatoes and potato

root eelworm, Het erodera rost ochiensis Woll.

BY GLYN L. JAMES" Imperial College Field Station, Ashurst Lodge, Sunninghill, Ascot, Berks.

(Received 16 February 1968)

SUMMARY

I n a study of the association between the causal fungus of brown root rot of tomatoes and Heterodera rostochiensis Woll., it was demonstrated that the nematode did not increase the susceptibility of the roots to invasion by the fungus; however, the fungus decreased the hatch of the potato root eleworm, the invasion of the host plant by the nematode, and number of new cysts sub- sequently produced.

I N T R O D U C T I O N

Dunn & Hughes (1964) investigated the interrelationships of potato root eelworms, Rhizoctonia solani and Colletotrichum atramentarium, on the growth of tomato plants. They found that combined inoculation with Heterodera rostochiensis and Rhizoctonia, Colletotrichum and Rhizoctonia, and with all three pathogens together checked growth significantly. Inoculation with Rhizoctonia alone caused a slight growth reduction, but Heterodera at the low level of inoculum used, alone or with Colletotrichum, or the latter pathogen alone, did not.

In addition to infections by the combination of pathogens tested by Dunn & Hughes, tomatoes are frequently attacked by potato root eelworms and a grey sterile fungus (GSF) : the latter pathogen was first recorded by Williams (1929). Termohlen (1962) found that GSF when attacking tomatoes caused the development of cortical root corkiness-one of the facets of brown root rot-an observation since confirmed in England by Preece (1964). Brown root rot debilitates but rarely kills the plant. Infected plants are shorter and, during sunny weather, have a tendency to wilt- indicative of a poor root system. The lower leaves are shed prematurely and yields often decreased appreciably (Last & Ebben, 1963, 1966).

Potato root eelworm was first recorded as a serious pest of tomatoes under glass in 1928 in Yorkshire (Thompson, 1949). It retards growth, the host becoming subject to wilting, with the lower leaves tending to die back (Southey, 1965). Feldmesser (1952) reported that, in the early stages of attack, tomato roots develop small swellings similar to the slight galling caused by minor infections of the root-knot nematode, Meloidogyne spp., an effect not noticeable on potatoes.

Rhodesia. * Present address : Rhodesia Sugar Association Experiment Station, P.O. Box 102, Chiredzi,

504 GLYN L. JAMES

To assess the interrelationships between potato root eelworm and GSF, common pathogenic inhabitants of the soil of commercial glasshouses used for tomato pro- duction, the following work was initiated.

T E C H N I Q U E S A N D B I O L O G I C A L M A T E R I A L

Production of sterile tomato seedlings Tomato seeds (variety Eurocross A) surface-sterilized in ‘ Milton ’, were germinated

on plates of malt extract agar, and transferred singly, if sterile, to prepared culture- tubes (Townshend, 1963) of sterile sand and nutrient solution (Hoagland & Arnon, 1950) subsequently being placed in illuminated test-tube racks. The nitrogen in the nutrient solution was in the form of ‘nitrate’ not ‘ammonium’, because Johnson & Townsend (1949) showed that free ammonia in ammonium carbonate inhibited hatch of Heterodera rostochiensis Woll. at zoo ppm. As tomatoes respond to the red end of the spectrum, Philips ‘Warm White’ tubes (TLAB4o W/29) were used. Each culture- tube was exposed to a 16 h photoperiod at a light intensity of 630 f.c. the developing roots being shielded, as only that part of the tube above the sand level protruded from a perforated wood spacing plate.

Preparation of GSF hyphal suspension Malt extract broth cultures of GSF were incubated for 2 weeks at 25 “C. The

mycelial mats were then separated and washed three times with 60 ml sterile water before being homogenized in an MSE homogenizer, at 14000 rev/min. The resulting suspension, in a further 60 ml of water, was checked for contaminants before being used experimentally.

Surface sterilization of potato root eelworm eggs and larvae Cysts, already soaked in water, were crushed on a channelled metal slide (Reid, 1955)

and washed into a macerator bottle. The suspension was then agitated, the macerator blades being replaced by rubber flails (Bijloo, 1954), for three periods each of 10 s, so separating most of the eggs and larvae from wall fragments of the cysts (Goodey 1963), which were removed by filtering through a nylon screen (92 meshes/in). The filtrate, containing eggs and larvae, was centrifuged at I 500 revlmin for 5 minutes, the sediment was resuspended in 5 ml and added to 25 ml of hydrogen peroxide-20-vol (Fenwick, 1956)-within a stoppered flask. The suspension of eggs and larvae in hydrogen peroxide was agitated on a wrist-action shaker for 2 h, poured into a capped sterile polypropylene tube and centrifuged at 1500 rev/min for 5 min. The supernatant liquid was removed using a sterile siphon tube to the residuum of 5 ml, then 20 ml of fresh sterile water were added. This washing process was repeated twice and, before estimating the concentrations of eggs and larvae and diluting appropriately with sterile water, the presence of contaminants was checked by incubating aliquots in thioglycollate broth.

Eaect of brown root rot of tomatoes on nematode invasion 505

E X P E R I M E N T A L

Experiments with on2y one variable ( I ) Ejjects of nematodes on root invasion by GSF

Two-week-old seedlings were inoculated with forty nematode eggs/g of sand and then incubated for a further fortnight when 4 m l of GSF hyphal suspension were added per culture-tube.

Plants were subsequently sampled at intervals of 2 ,4 ,8 and 16 days, when colonizing populations of GSF were estimated. Roots were surface-sterilized by first immersing in 75 yo ethyl alcohol for 30 s, followed by 0-1 yo mercuric chloride for I min, washed five times in sterile water, and then homogenized in small sterile masticator bottles containing 5 ml of sterile water; I ml aliquots of suspension were pipetted into Petri dishes to which were added, with thorough mixing, 20 ml of malt extract agar and Rose Bengal (0.004%) precooled to c. 40 "C. After a week's incubation it was found that numbers of GSF colonies, which increased as the interval between plant inocula- tion and sampling increased, were unaffected by nematodes (Table I).

Table I . The development of GSF on tomato roots previously inoculated with potato root eelworms

Roots were sampled at different intervals after GSF inoculation when colonies isolated from each root system were counted.

Roots inoculated with Roots sampled, -7 r h

days after GSF Nematodes and inoculation GSF GSF

2

4 8

16

0 0 0.6 04 f 0 2 3.8 3.6 rfr 0.3 15'2 11.6+1.9

Table 2. Effects of previous inoculation with GSF on subsequent tomato root invasion by potato root eelworms, numbers of invading eelworms per root being counted

Roots sampled, < * >

inoculation GSF Nematodes

Roots inoculated with

days aftcr nematode Nematodes and

2 4 8

16

0 0.4 0.6 0.8

4.8 If: 0.7 I O . O + 1.6 28.2 f 44 2'2

(2) Ejjects of pre-inoculating with GSF on the subsequent eelworm invasion of tomato roots One-week-old seedlings were inoculated with 4 mi of GSF hyphal suspension per

tube and then incubated for a fortnight before culture-tubes were infested with forty nematode eggs/g of sand.

After the appropriate time interval the roots were stained in 0.05 % cotton blue in lactophenol for 15 min at IOO "C, then washed in water, and differentiated in plain lactophenol for 12 h.

GLYN L. JAMES

A comparison of the roots with and without GSF showed that the fungus decreased numbers of invading nematodes from 28.2 to 2.2 per root system 16 days after eggs were added (Table 2).

(3) E'ects of simultaneous^ inoculating tomatoes with GSF andpotato root eelworms on the numbers of root-invading nematodes

Seedlings, after growing in a mixture of sand, brickdust and pulverized peat (I I : 5 : I) for approximately a month, were transferred to small plastic pots containing 150 g of this mixture plus fifty Heterodera rostochiensis cysts (c. fifty eggslg). Simulta- neously GSF was added to half the pots, but in this instance, in contrast to earlier experiments, the fungus was cultured in flasks on 20 g vermiculite (10-14 mesh/in) which, having previously been soaked in 80 ml of malt extract broth, was autoclaved at 115 "C for 10 min. After 2 weeks incubation with intermittent shaking, the vermi- culite was washed free of residual malt extract and I g added per 10 g of sand, brick- dust and peat mixture ; uncontaminated vermiculite was added to the series without GSF.

Simultaneously inoculating with GSF decreased numbers of invading eelworms from 1850 to 680 ( k 178.5) when assessed 16 days after eelworms were added. Irrespec- tive of the presence or absence of GSF, numbers of eelworms subsequently decreased (Table 3), probably because (a) swollen females that had burst through the root cortex were easily dislodged when roots were washed, and (b ) many males had by now migrated from root tissues.

Table 3. EfSect of GSF on the invasion of tomato seedlings by potato root eelworm larvae: number of nematodes per g fresh weight of roots

(days) GSF Nematodes Sampling time Nematodes and

4 8

16 32

0

360 680 290

0

775 f 95'2 18505 178.5 540 * 37'4

Factorial experiments (I) EfSects of d#erent amounts of GSF and nematode inocula

Two-week-old seedlings in culture-tubes were inoculated with the factorial com- binations of 0, 0.5, 1.0, 2.0 or 4.0 ml of GSF hyphal suspension per tube, and 0, 10, 20,40 or 80 nematodes eggs/g of sand. When the inocula had been added, the volumes of the liquid pipetted into each tube were adjusted to 5 ml so that the water status was the same in each. Each factorial combination was replicated four times; after a month two were used for fungal estimates and two for nematode counts.

Brown root rot symptoms were scored visually on an arbitrary scale 0-5, and these values were found to be related to the numbers of GSF colonies developed after I week on malt agar from roots that had been surface-sterilized and homogenized (Fig. I).

The remaining seedlings were stained in 0.05 yo cotton blue/lactophenol and num- bers of nematodes counted (Table 4).

EfJect of brown root rot o j tomatoes on nematode invasion 507 As in earlier experiments GSF decreased numbers of invading nematodes from

2.8 to 0.0 per root with 0.0 and 2.0 g of GSF inoculum respectively. The overall level of eelworm invasion is low and probably correlated with the lower rate of growth of plants growing in culture-tubes compared with those exposed to external atmospheres. Gibson (1967) found that carbon dioxide diffusion into such tubes limited photo- synthesis, and this nematodes.

is possibly reflected in plants being less attractive to invading

I,,,,,,,,,,,,,,,, 2 4 6 a 10 12 14 16 l a 20 22 24 26 2a 30

Number of GSF colonies

Fig. I. Relationship between the two methods of assessment of brown root rot.

Table 4. Eflect of various combinations of GSF and potato root eelworms at daTeerent levels on the invasion by Heterodera of the tomato roots

Nematode GSF hyphal suspensions

1. I 3

eggslg 0 0 0.5 1'0 2'0 40 Mean

1 0 I 0 . 5 0 0 0 0.3 20 2 2'5 0 0 0 0 9

0 0 0 0 0 0 0

40 3 2 3 0 0 1.6 80 8 5 2 0 0 3'0

Mean 2.8 2'0 1'0 0 0

( 2 ) Effects of darerent dates of inoculating tomatoes with nematodes and GSF Seedlings, I month old, grown in a sterilized 3 : 2, loam: sand, mixture, were trans-

ferred with their soil ball from small plastic pots (5-6 cm) to larger earthenware pots holding 5 kg of soil. A week later the following series of treatments was initiated: ( a ) pots were infested with nematodes and 2 weeks later with GSF; (b ) pots were infested with GSF and with nematodes 2 weeks later ; (c ) pots infested with nematodes and GSF simultaneously; ( d ) uninfested controls.

The nematode inoculum was added at a rate of forty nematode eggs/g of soil, and GSF as a hyphal suspension prepared from week-old cultures grown on malt extract broth, at a rate of five cultures per pot. As expected, the progress of GSF attack was unaffected by nematodes, but when soil was extracted, 8 weeks after treatments were initiated, more cysts occurred where seedlings had been pre-inoculated with GSF,

508 GLYN L. JAMES

641200 g of soil compared with c. 36 (Table 5). This result contrasts with those ob- tained in earlier experiments, but may be attributed to the fact that many larvae unsuccessfully competed for the relatively few feeding sites available when nematode inoculum was added to very small seedlings in treatments (a) and (c) . Two weeks later, when nematodes were added to treatment (b), seedlings were appreciably larger. Nonetheless when the seedlings were simultaneously inoculated with nematodes and GSF, numbers of eggs/cyst were decreased from 87/100 to 41.

No differences in yield, root length and fresh weight could be detected after 8 weeks as a result of the timing of the inoculations.

When this experiment was repeated in culture-tubes using 4 m l of GSF hyphal suspension and/or 1200 nematode eggs (forty eggs/g) as inocula, more larvae invaded roots when nematode inocula were added before GSF, numbers of larvaelroot in- creasing from 2-4 to 11.0 (Table 6).

Table 5 . Effect of dzjferent times of inoculating GSF and Heterodera rostochiensis on the jinal nematode population

Treatments Cysts per zoog Eggslcyst Eggslg Nematode before GSF 38 87 16.5 GSF before nematode 64 I00 32.0 Both pathogens together 34 41 7'0 S.E. of mean f 5'3 5 11.5 k 5'0

Table 6. Eflect of dzjferent times of inoculating GSF and Heterodera rostochiensis on the nematode invasion of tomato seedlings

Treatments No. of nematodes/g root

Nematode before GSF 11'0

GSF before nematode 9'4 Both pathogens together 2'4 S.E. of mean a 1'5

D I S C U S S I O N A N D C O N C L U S I O N S

The genera Meloidogyne Goeldi, 1887, and Heterodera Schmidt, 1871, belong to the same family, Heteroderidae Skarbilovich, I 947 ; however, the associations of many members of the former genus with both fungi and bacteria causing plant diseases have been extensively reported, whilst similar information about Heterodera is scanty.

These experiments have shown that the invasion of the tomato roots by potato root eelworm did not predispose the host plant to attack by the causal fungus of brown root rot. Graham (1966) has suggested that, in commercial glasshouse soils used conti- nuously for the production of tomatoes, where both GSF and Heterodera rostochiensis are present, the nematode population does not build up as expected; a fact confirmed by James & Hague (1967) when methyl bromide was used to fumigate infested soil. Where root rots are not of importance to tomato growers, for instance in the south- west of England, then potato root eelworm populations build up in the soil and have a detrimental effect on tomato yield.

Effect of brown root rot of tomatoes on nematode invasion 509 James (1966) has shown that GSF produces a factor in liquid culture inhibitory to

the hatch of Heterodera rostochiensis. The presence of such a substance in the soil would explain the low pathogenicity of the potato root eelworm towards the host plant, and possibly it is only when GSF is actively colonizing the roots of tomato plants that the substance causing the reduction in hatch is produced in quantity.

In nematode/fungal interractions Pitcher (1965) noted that the obligate plant- parasitic nematode would appear to be especially vulnerable to competition when the two types of pathogen compete for nourishment from the same individual host plant. Christie & Perry (1959) refer in somewhat general terms to the incompatibility of plant-parasitic nematodes and many decay-promoting organisms, and Du Charme (1958) found that secondary fungal invaders diminished the severity of damage caused by Radopholus sirnilis in the roots of citrus. The microbial associates of root disease fungi, which abound in the soil, are recognized to have a marked influence on them, and in turn on the development of the diseases they cause (Henry, 1961), the most pronounced and common effect being antagonistic or deterimental (Dobbs & Hinson I 953). Were it not for this widespread fungistatic and/or fungicidal action root diseases would be more destructive than they are.

Pitcher (1965) postulates that some nematodes may have an antibiotic mechanism which helps to keep competitors at bay, leaving them ‘to enjoy the fruits of their enzymes in peace’. It would appear from the results reported here that root-infecting fungi may have corresponding substances which enhance their success in infecting host tissue by inhibiting nematodes.

My thanks are due to the late Professor B. G. Peters and Dr N. G. M. Hague for their continued interest and guidance during my Ph.D. studies, of which this work is a part.

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