Stimulation of respiratory pathways in tomato roots infested by Meloidogyne incognita

Physiological and Molecular Plant Pathology (1987) 30,461-466

Stimulation of respiratory pathways in tomato roots infested by Meloidogyne incognita G . ZACHEO

and T .

BLEVE-ZACHEO

Istituto di .Nematologia Agraria Applicata ai Vegetali, C.N.R., Via Amendola 165/A, 70126 Bari, Italy (acceptedfor publication October 1986)

Respiration of tomato roots susceptible and resistant to Meloidogyne incognita was measured during infestation. No significant changes in respiratory rate occurred in susceptible tomato roots, during infestation by M. incognita . In resistant tomato roots, a pronounced increase of both cyanidesensitive and cyanide-resistant oxidases, was observed during nematode attack . The time-course of the respiration during 12 days, after nematode inoculation, showed that resistant tomato roots responded with a rapid increase in cyanide-sensitive and cyanide-resistant respiration as invading nematodes progressed; no changes were observed in the susceptible tomato roots . Change in the rate of oxygen uptake paralleled an increase in nematode density in resistant tomato roots ; oxygen uptake rose linearly to an infestation level of 50 juveniles for each seedling, above which value it declined . The physiological significance of the alternative respiratory pathway is discussed .

INTRODUCTION

It is commonly observed that when susceptible plants become infected by pathogens there is a marked increase in the rate of respiration . This subject has been extensively studied in a wide variety of plants infected by pathogenic micro-organisms and is well documented [12] . In contrast, the reports on the respiratory changes in resistant plants challenged by pathogens are inconsistent [12] . Millerd & Scott [9] found that a barley cultivar highly resistant to powdery mildew reacted with a more rapid rise in respiration than did a susceptible cultivar, whilst another resistant cultivar reacted to inoculation with a decrease in respiration . Where a high rate of respiration follows pathogen attack on resistant plants it is generally assumed to be associated with biochemical and structural changes accompanying the hypersensitive response in the host plants [4, 7] . Increased rates of respiration in infected tissue are accompanied by increased levels of oxidases, such as polyphenol oxidase and ascorbic acid oxidase, sufficient to account for extra electron flow in the respiration [1] . However it is difficult to accept that these increases are sufficient to explain all the rise of respiration since each of these enzymes are sensitive to inhibition by cyanide, and it has been reported that the rise in respiration in infected plants is frequently associated with the operation of terminal oxidases that are resistant to inhibition by cyanide and antimycin A [2,11, 13] . The presence of a cyanide-resistant electron transport pathway has been reported in excised roots of many higher plants [6, 8, 14] . Janes & Chin [5] have reported the presence of multiple oxidases in excised 0885-5765/87/030461 +06 $03 .00/0

© 1987 Academic Press Inc . (London) Limited



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G . Zacheo and T. Bleve-Zacheo

tomato roots, including the classical cytochrome oxidase, inhibited by 10 -4 M cyanide, a second, less cyanide-sensitive oxidase requiring 10 -3 M cyanide for inhibition and a cyanide resistant oxidase . In the literature there appears to be only one reference to the behaviour of the respiratory rate in plants attacked by nematodes, which shows that the respiration rate of galls induced by a Meloidogyne sp . in susceptible tomato roots was no greater than that for ajacent tissue or uninfected roots [3] . In this paper we present data to show the effect of Meloidogyne incognita (Kofoid & White) Chitw . on the cyanide sensitive and cyanide resistant respiratory pathways in roots from resistant and susceptible strains of tomato . MATERIALS AND METHODS

Seedlings of tomato (Lycopersicon esculentum Mill .) cultivars Roma VF, Early Pack and Red Stone (susceptible) and VFN 8, Piersol and Rossol (resistant) were germinated in sterilized quartz sand . Uniformly germinated seeds were transferred into 3-cm clay pots containing quartz sand and divided into two groups, one uninfested and used as the control, and the other immediately inoculated with active juveniles of M. incognita race 2 . The methods used to measure root respiration were the same as those employed by Lambers & Van De Dijk [8], using an oxygen electrode (Rank, Bottisham, Cambridge, U .K .) . Intact roots (0 . 1 g fresh weight), excised from the shoots, were placed under vacuum in 0. 1 M KH 2 PO4i pH 7, for 5 min, in order to eliminate air present in the cavities; this was especially important in tissues infested by nematodes . Roots were subsequently transferred to fresh buffer for 20 min, before respiration was measured in 1 ml of the same buffer, at 25 ° C . Values for the following were determined : (1) Vey, ( cytochrome oxidase=cyanide-sensitive respiration), the difference between the VV (total respiration) rate and that obtained in the presence of 1 mm KCN (sufficient to cause maximal inhibition of the cytochrome oxidase) ; (2) Ve,t (alternative oxidase = cyanide-resistant respiration) was the difference between the respiration rate in the presence of 1 mm KCN and 1 mm salicylhydroxamic acid (sufficient to cause maximal inhibition of the alternative oxidase) ; (3) Vres , that rate of oxygen uptake observed in the presence of both 1 mm KCN and 1 mm salicylhydroxamic acid . In this paper, Vt = Vey,+ V,,, t. All values are the means of four experiments and each experiment employed three or four replicates . After the respiration measurements the roots were dried overnight at 70 ° C and weighed . To assess penetration by nematodes, roots were stained with lactophenol and fuchsin and observed under a light microscope . RESULTS

Table 1 shows data concerning the number of juveniles and the rate of multiplication of nematodes (number of females) associated with the different cultivars of tomatoes used . These can obviously be divided into susceptible and resistant cultivars . In the susceptible cultivars the juveniles developed into female nematodes after 30 days, whilst in the resistant cultivars no females were detected .



Respiratory pathways in M.

Incognita

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TABLE I

Multiplication (No . of nematodes g` roots) of Meloidogyne incognita in susceptible (S) and resistant (R) tomato roots 30 days after inoculation with 50 juveniles per seedling'

Cultivar

Juveniles

Adults

Roma VF (S) Early pack (S) Red stone (S) Rossol (R) Piersol (R) VFN 8 (R)

110±29 204±21 101+13 50± 14 80±12 64± 13

1349+192 913+159 842±97 0 2 2

Values are means of 10 replicates±SD .

TABLE 2

Root respiration, V,,,, and V, t,(nmol 0 2 min - ' mg -1 dry wt) and number of Meloidogyne incognita juveniles which have penetrated in susceptible (S) and resistant (R) tomato cultivars'

Healthy

Infested

Healthy

Infested

No . of juveniles penetrated each seedling

1 . 57±0 . 17 2 . 22±0.41 2 .46±0 . 41 1 . 89±0 .43 2 . 55±0 .31 2 . 22±0 . 30

1 . 50±0 . 18 2 . 17±0 . 49 3 . 03±0 . 14 4 . 00±0 .63 4 . 02±0 . 59 3 . 95±0 .89

1 . 31±0. 11 1 . 42±0 . 11 1 . 71±0. 09 1 . 79±0.37 2 . 29±0. 43 2 . 40±0. 27

0. 89±0. 14 1-21+0-10 1 .61±0 . 13 3 . 98±0 .45 3 . 90±0 . 28 3 . 50±0 . 33

36. 50±5 . 57 29. 72±7 . 13 31 .00±3 . 91 10 .00±3 . 16 17 . 25±3 . 86 12 . 50±2 . 64

VV ,,, Cultivar Roma VF (S) Early pack (S) Red stone (S) Rossol (R) Piersol (R) VFN 8 (R)

V,,,

'Fifty juveniles of M . incognita were added to each tomato seedling and the respiration was measured 6 days later; V., and V,,, were measured as described in Materials and Methods . Values are means of four experiments ± SD .

Table 2 shows the influence of nematode infestation on the relative contribution of the cytochrome oxidase (VCy1 ), and the alternative cyanide-resistant oxidase (V est ), to the overall respiration rate (V,), 6 days after infection . In the susceptible cultivars, infestation did not result in any major change in respiratory rate via either oxidase, whilst in resistant cultivars electron transport through both pathways very nearly doubled . There were fewer juvenile nematodes in the resistant than in the susceptible cultivars (Table 2) . From these results it is apparent that the Roma and Rossol cultivars represented the extremes of susceptibility and resistance, respectively, and consequently were selected for further study . The time-course of respiration changes in healthy and infested cultivars is presented in Fig . 1 . In susceptible cultivars [Fig . 1(a), Roma], respiration via the cytochrome chain and the alternative oxidase declined four days after nematode inoculation ; whilst in resistant cultivars [Fig . 1(b), Rossol] respiration via both oxidases was



464

G . Zacheo and T. Bleve-Zacheo

a T I~

E TC E ô 2 o E

r t.

C

I

I I I I I I I I I 1 0 2 4 6 8 10 12 2 4 6 8 10 12 0 Time after infestation (days) FIG. I . Changes of VV,., and V. 1, and juvenile penetrations during disease development in susceptible Roma VF (a) and resistant Rossol (b) tomato roots . Inoculum concentration was 50 juveniles per seedling . Vertical bars represent standard deviations of four experiments . (], O, VVy, and V.,,, respectively, in uninfested tomato roots ; ∎, •, V.., and V.,,, respectively, in infested tomato roots . z~,, Number of penetrated juveniles, regression lines = (a)y=4 •I x-2 . 56, r=0 .99 ; (b) y= 1-53x+ 0 . 52, r=0 .97 .

initially, markedly stimulated and then decreased . Figure 1 also shows the number of juvenile nematodes recovered from the roots . In the case of the susceptible cultivar, Roma, there was a linear rate of increase over the experimental period ; in the case of the resistant cultivar Rossol, the number of nematodes also increased linearly but at a lower rate . Subsequent investigation of the relationship between the level of infestation and the magnitude of the respiratory response of the resistant cv . Rossol, was undertaken over the first 6 days and the data obtained are given in Fig . 2 . These show that the level of nematode infestation was paralleled by a rise in respiration rate for inoculation up to a level of 50 juveniles per plant ; above this value the degree of stimulation of oxygen consumption decreased .

DISCUSSION

Data obtained using the susceptible cultivar Roma showed only a slight increase in the rate of respiration during the first 4 days after inoculation (cf. Bird & Millerd [3]) with M. javanica . When nematodes invade susceptible cultivars they induce galls composed of giant cells which contain more protein and nucleic acid than normal cells [10] . Because such cells would be expected to be characterized by a high biosynthetic activity it would be reasonable to assume that the respiration of the galls would be higher than uninfected tissue . It is surprising that this is not reflected in the data obtained . In contrast, when using the resistant cultivar, Rossol, the electron flux through both the cyanide-sensitive and cyanide-resistant pathway was markedly increased for a longer period of time following inoculation with M. incognita . It is also clear from Fig . 2 that there was a relationship between the level of infestation and the magnitude of the



Respiratory pathways in

M. Incognita

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I I I I I 20 40 60 80 No . of inoculated juveniles per plant

100

FIG . 2 . Respiration (∎) of whole tomato roots of the resistant cultivar Rossol, reacting hypersensitively in relation to the population density of Meloidogyne incognita . V = V.Y, + V.,,. Each point is the mean value for four experiments, vertical bars indicate standard deviations . L, Number of juveniles penetrated 6 days after inoculation :y=0 . 36x+0 . 52, r=0. 99 .

increase in respiration . The increase in respiration in resistant plants must be considered in relation to the hypersensitive response . This response is developed in a limited number of host cells which are killed quickly and the pathogen contained in a localized necrotic lesion . Hypersensitivity can be viewed as extreme cellular reactivity which may confer a high degree of disease resistance to the whole plant [16] . Such changes are accompanied by increased activities of many oxidative enzymes (including polyphenol oxidases, ascorbic acid oxidase and peroxidases) and alternative cyanide-resistant respiration . Polyphenol oxidase and ascorbic acid oxidase could function as cyanide-sensitive terminal oxidases contributing to the overall respiratory stimulation . Whilst the peroxidases and polyphenol oxidases could be involved in the oxidation and polymerization of phenols to produce browning and formation of lignin-like barrier substances [15], it is also possible that these oxidative enzymes may give rise to a range of free radicals, including those of oxygen, that could contribute to the phytotoxic effect of nematode infestation . Further evidence concerning the mechanism involved in mediating the cyanide resistant respiratory chain is essential before it is possible to appreciate fully its role in the hypersensitive response to nematode attack . The authors wish to thank Professor J . M . Palmer, Imperial College, London, for critical comments and suggestions on the manuscript . Research work supported by C .N .R ., Italy . Special grant I .P .R .A ., sub-project 1 . Paper No . 712 . REFERENCES R. & CALABRESE, G . (1977) . The increase ofhydroxyprolinecontaining proteins in Jerusalem artichoke mitochondria during the development of cyanide-insensitive respiration . Biochemical & Biophysical Research Communications 74, 1637-1641 .

1 . ARRIGONI, O., DE SANTIS, A ., ARRIGONI-LISO,



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2 . BENDALL, D . S . & BONNER, W . D . (1971) . Cyanide-insensitive respiration in plant mitochondria . Plant Physiology 47, 236-245 . 3 . BIRD, A . F . & MILLERD, A . (1962) . Respiration studies on Meloidogyne induced galls in tomato roots . Nematologica 8, 261-266 . 4 . DEVERALL, B. J . (1977) . Defence Mechanisms ofPlants . Cambridge University Press. Cambridge . 5 . JANES, H . W . & CHIN, C . (1981) . The effect of age and growing conditions on cyanide resistance in cultured tomato roots . Plant Science Letters 23, 307-313 . 6 . KANO, H . & KAGEYAMA, M . (1977) . Effects of cyanide on the respiration of muskmelon . (Cucumis melo L .) roots . Plant and Cell Physiology 18, 1149-1153 . 7 . Kuc, J . A . (1972) . Phytoalexins . Annual Review of Phytopathology 10, 207-232 . 8 . LAMBERS, H . & VAN DE DIJK, S . J . (1979) . Cyanide-resistant root respiration and tap root formation in two subspecies of Hypochaeris radicata. Physiologia Plantarum 45, 235-239 . 9 . MILLERD, A . & SCOTT, K . (1956) . Host pathogen relations in powdery mildew of barley. II . Changes in respiratory pattern . Australian Journal of Biological Science 9, 3714 . 10 . OWENS, R . G . & NOVOTNY, H . M . (1960) . Physiological and biochemical studies on nematode galls . Phytopathology 50, 650. 11 . PALMER, J . M . (1976) . The organization and regulation of electron transport in plant mitochondria . Annual Review ofPlant Physiology 27, 133-157 . 12 . SMEDEGAARD-PETERSEN, V . (1982) . The effect of defence reactions on the energy balance and yield of resistant plants . In Active Defense Mechanisms in Plants, Ed . by R . K. S . Wood, pp . 299-315 . Plenum Press, London . 13 . SoLoMos, T . (1977) . Cyanide resistant respiration in higher plants . Annual Review of Plant Physiology 28, 279-297 . 14 . VAN DER PLAS, L . H . W ., SCHOENMAKER, G. S . & GERBRANDY, S . J . (1977) . CN resistant respiration in a Convolvulus arvensis L . cell culture . Plant Science Letters 8, 31-33 . 15 . VAN LOON, L . C . (1982) . Regulation of changes in proteins and enzymes associated with active defence against virus infection . In Active Defense Mechanisms in Plants, Ed . by R . K . S . Wood, pp. 247-273 . Plenum Press, New York . 16 . WHEELER, H . (1975) . Plant Pathogenesis . Springer-Verlag, Berlin .