The relationship between infections of the cotyledons of Gossypium barbadense and G. hirsutum with Alternaria macrospora and cotyledon abscission

293

Physiological and Molecular Plant Pathology (1989) 35, 29 3-299

The relationship between infections of the cotyledons of

Gossypium barbadense and G. hirsutum with Alternaria macrospora and cotyledon abscission B . SPROSS -BLICKLE,t, J . ROTEM,+ M . PERL+ and J . KRANZ t Tropeninstitut, University of Giessen, Giessen 6300, Federal Republic of Germany ; + Agricultural Research Organization, The Volcani Center, Bet Dagan 50250 Israel (Accepted for publication March 1989)

The rate at which cotyledons of the cotton species Gossypium barbadense, cv . Pima, infected with Alternaria macrospora abscissed was related to disease severity . Shedding was increased by cultivating the plants at high temperatures prior to inoculation, under poor nutritional conditions, or by inoculating the cotyledons at an early stage of development . Abscission of infected cotyledons by the relatively resistant cotton species G . hirsutum cv . Acala required about double the time required by the susceptible G. barbadense . Abscission also occurred in both species in response to infection with Alternaria alternata, and in response to injection with cell-free extracts of pathogenic Alternaria spp . and non pathogenic Fusarium and Botrytis spp . It also occurred in response to injection with chemicals, to mechanical injuries, heat shock or growth in continuous darkness . Premature abscission appears to be a n .on-specific response to stress, but in the field, abscission of leaves by G . barbadense cv. Pima infected with A . macrospora appears to be a response to the infection rather than to any other stress agent .

INTRODUCTION

Essentially, defoliation is a natural process associated with leaf senescence . Factors that lead to defoliation have been described in numerous publications [1, 6, 11] . The premature abscission of infected leaves has been observed in a number of host-pathogen systems [5, 7, 14], and attributed to the action of ethylene produced by the host [4, 13] or the pathogen [12] . Premature leaf shedding in response to infection is rare in the relatively resistant cotton species Gossyßium hirsutum L ., cv . Acala . In the susceptible cotton species Gossypium barbadense L ., cv . Pima, infection by A . macrospora Zimm . causes premature shedding resulting in reductions in yield [3] . The rate of shedding of true leaves in G . barbadense cv . Pima was found to be correlated with the rate of disease development [2], but this investigation was too limited to determine if infection was the only factor involved . It was not clear if shedding was directly associated with the A . macrospora infection or if it was influenced by the host's propensity to shed . To clarify these questions we studied the effects on abscission of some parasite and host factors, and of some chemical and physical agents .

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MATERIALS AND METHODS Plant materials

Cotton plants of the species Gossypium barbadense, cv. Pima S-5, and G . hirsutum cv . Acala were grown in 0 . 3 1 pots filled with a mixture of sandy loam, peat and sand, (2 : 1 :1, v : v : v) fertilized every fortnight with 0. 5 g of NPK (1 : 1 : l ,v : v : v) with microelements (standard soil) . The plants were kept in a growth room at 25 °C, in a relative humidity of 50-70 °,o, and a 12 h photoperiod provided by Sylvania Gro-lux lamps giving a light intensity of 120 tE cm -2 sec' . All experiments used ten plants per treatment . Fungus culture and inoculations Isolates of Alternaria macrospora and A . alternata (Fr .) Keissler were obtained from

infected plants growing in a commercial cotton field . Other fungi used in this work were Fusarium oxysporum f. sp . lycopersici S . & H . and Botrytis cinerea Fr . All of them were maintained in culture on V-8 agar in Petri dishes . A spore inoculum was applied to cotyledons of 14-16-day-old plants by means of Schein's quantitative inoculator [9] or in filter paper discs . Assessment of disease

Disease severity was assessed visually as the percentage of the area of inoculated cotyledons which became necrotic . The effect of nutrition and post-inoculation temperature

Interaction between nutrition and post-inoculation temperature was tested in G . barbadense cv . Pima using plants grown in the fertilized standard soil and in a nonfertilized, poor soil consisting of a mixture of sandy loam and vermiculite, 1 :4 (v : v) . The plants were incubated for 24 h after inoculation in a dew chamber and then transferred to a growth room at 25 or 35 ° C . The effect of inoculation with pathogenic and non-pathogenic fungi and of treatment with cell free extracts of the fungi or chemicals on cotyledon abscission

Cotyledons were inoculated with spore suspensions of the pathogenic species A . macrospora and A . alternata or of the non-pathogenic species Fusarium oxysporum f. sp . lycopersici or of Botrytis cinerea as detailed in the Results Section ; or were injected, using a hypodermic needle, with 0.3 ml of distilled water containing fungal spores or cell-free extracts of the fungus . The extracts were obtained from 11-day-old cultures in which the fungus was grown over a cellophane film 8 cm in diameter . The cellophane, with mycelium and spores, was removed and homogenized with a mortar and pestle in the presence of glass beads (60 mesh) . Then, 50 ml of water was added and the mixture homogenized for 3-5 min . The homogenate was filtered through two layers of gauze, centrifuged at 5000 g for 10 min, and filtered twice through Whatman No 1 filter paper to produce a filtrate which was cell-free, as determined by microscopic examination and growth on Petri dish media . Extracts used for injection contained 1 . 5 mg protein . Sufficient solutions of chemicals were injected using a hypodermic syringe, for the injected materials to spread throughout an area of 5-7 cm 2 within the cotyledon tissue . The effects on shedding of physical treatments included, mechanical damage, heat



295 Cotyledons of G. barbadense and G. hirsutum . The controls were untreated plants with cotyledons shock and the continuous darkness injected with distilled water . RESULTS Relation of disease severity to cotyledon abscission

The relationship between disease severity and cotyledon abscission in the susceptible G . barbadense cv . Pima was tested . A range of levels of infection was obtained by inoculating cotyledons on a target of 4. 9 cm 2 with a spore density of 6, 26 or 86 spores cm 2 , using Schein's inoculator . The inoculated plants were kept wet (wetness period) for 6, 12 or 24 h at a temperature of 15, 25 or 35 ° C after inoculation . At the end of the treatment the plants were transferred to 25 ° C and incubated at this temperature until abscission . (a) WP :6h

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Temperature (°C) Fir- 1 . Relation between disease severity (a) and cotyledon shedding (b) induced by Alternaria macrospora infections of cotyledons in cotton G . barbadense cv . Pima at different periods of leaf surface wetness . Various levels of disease were induced by varying the inoculum level applied to 4 . 9 cm 2 of cotyledon surface . The inoculum level was (--- • •- -) six spores, (----) 26 spores, and (-- ) 86 spores per cm -2 of cotyledon . Duration of leaf surface wetness (WP) varied from 6-24 h. Disease severity (a) is expressed as per cent of necrotic surface on the cotyledons 15 days after inoculation . Rate of shedding (b) is expressed as the number of days between treatment and cotyledon abscission . Parameters are plotted as means+ SE .

Figure 1 (a) shows that at all temperatures and for all durations of wetness period, the highest level of disease was induced by inoculation with the largest inoculum . A temperature of 25 ° C was found to be optimal at all combinations of inoculum size and wetness period . A period of wetness of 24 h was optimal for all combinations of temperature and inoculum size .



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Figure 1 (b) records the number of days between inoculation and cotyledon abscission . Comparison with Fig . 1 (a) shows that in most plants the greater the disease severity, the earlier the cotyledons abscissed . Furthermore, the temperature, wetness period and inoculum size which were optimal for disease development were associated with the earliest shedding . The cotyledons on the healthy controls abscissed 37 ± 2 days after inoculation of the other plants . Effect of temperature of plant cultivation before inoculation Seeds of G . barbadense cv . Pima were germinated at 25 ° C and the seedlings then grown at 15, 25 or 35 ° C for 10, 15, 20, 25 or 30 days until inoculation . An inoculum, of 429±43 spores was applied to each cotyledon over an area of 3 . 14 cm2 using a Schein's quantitative inoculator . After inoculation the plants were incubated for 24 h in a dew chamber, at 20 ° C, and then transferred to a growth chamber at 25 ° C until the cotyledons abscissed . Uninoculated seedlings grown at 35 °C grew faster and their cotyledons abscissed earlier (after 52±3 days) than plants grown at 15 ° C (after 78±2 days) . Plants grown at 35 °C before inoculation developed more severe disease by the eleventh day after inoculation if inoculated 10 days after germination (63±3%) than if inoculated 30 days after germination (29±4%) . The inoculated cotyledons also abscissed earlier from the earlier (25±2 days after germination) than the later inoculated plants (45± 5 days after germination) . In contrast, plants grown at 15 ° C before inoculation developed less disease overall viz . 33+3% on the plants inoculated 10 days after germination and 2 % on the plants inoculated 30 days after germination . The cotyledons on the earlier inoculated plants abscissed 36±3 days after germination on the later inoculated plants 61 ± 5 days after germination . Effects of nutrition and post-inoculation temperature on cotyledon abscission by G . barbadense Cotyledons of healthy control plants grown in the fertile standard soil abscissed 57±2 days after germination when grown at 25 ° C and 56±2 days after germination when grown at 35 ° C . Shedding of healthy cotyledons was speeded up by cultivation in nutrient-deficient soil and occurred 50± 1 days after germination at 25 ° C, and 50±3 days after germination at 35 ° C . In this experiment cotyledons were inoculated by applying filter paper discs (5 mm in diameter) dipped in a spore suspension containing 9 x 10 3 spores ml -1 . Cotyledons of plants incubated at 25 °C in the standard soil attained disease severity of 96 % and in nutrient-deficient soil a disease severity of 66 and were shed 36± 1 and 26± 1 days after germination, respectively . These differences were significant at the 5 % level . Inoculated cotyledons of plants incubated at 35 ° C reached a maximum disease severity of about I % and shed at the same time as those of healthy plants . Relationship between disease severity and abscission by G . barbadense and G. hirsutum Shedding in the sensitive G . barbadense cv . Pima was compared with that of the relatively resistant G . hirsutum cv . Acala using seedlings inoculated by applying 5 cm 2 filter paper discs dipped in a spore suspension containing 9 x 10 4 spores ml -1 . The maximum disease severity attained on G . barbadense was 75 ± 3 % and on G . hirsutum was 56 ±4% . The infected cotyledons of G . barbadense shed 26 ±1 days after germination



Cotyledons of G.

297 . The cotyledons of healthy and those of G. hirsutum shed 45 ± 2 days after germination control plants of both Gossypium spp . abscissed 34± 1 and 66±4 days after germination, respectively . barbadense

and G . hirsutum

The effect of infection by A . macrospora and of damage by chemical and physical agents on abscission by G . barbadense and G . hirsutum An area of 5 cm 2 of the cotyledons of G . barbadense and G . hirsutum was either inoculated

with A . macrospora, damaged by puncturing with a needle, rubbed with carborundum, injected with cell-free extracts of A . macrospora, distilled water or solutions of various chemicals . Additional treatments included immersion of leaves in hot water of 60 ° C for 5 sec or wrapping the cotyledons in aluminium foil to exclude the light for 1 week . Infection with A . macrospora resulted in a disease severity of 86% in G. barbadense and in 3 % G . hirsutum . The results are given in Table 1 . Abscission was not hastened in TABLE I

The effect of pathogenic factors and chemical and physical injuries on the shedding of cotyledons by G . barbadense and G . hirsutum .

Cotyledon treatment

Shedding in G . barbadense

Untreated (control) Injected with 0. 3 ml distilled water Inoculated with Alternaria macrospora° Densely punctured with needle Damaged by rubbing with carborundum Half-cotyledon dipped 5 sec in hot (60 °C) water Wrapped in aluminium foil and kept 1 week in dark Injected with A . macrospora extract (1 . 5 mg protein) Injected with 0. 075 mg fentin acetate Injected with 1% NaCl

25±1 25±2 10±0 .5* 20 ± 1 21± 1 * 18± 1* 14±2* 20+2* 8±1* 6±1*

G . hirsutum

49+3 41±2 46±3 35±3* 36±3* 39±2* 15±3* 31 ± 2* 26±3* 7±2*

'Days after treatment, means±SE . Values marked with an asterisk are significantly different from the controls at 5 % level . 'The cotyledons were inoculated by applying a 5 cm' filter paper containing 460 spores . The inoculated plants were transferred to a humidity chamber for 24 h and then incubated in a growth chamber at 25 ° C approx . 9 x 103 spores ml - ' and of F. oxysporum and B. cinerea approx . 20 x 10 3 m1 - ' . 'All injections were made in a volume of 0 .3 ml distilled water .

either species by injection with distilled water or in G . hirsutum by infection with A . macrospora ; but was significantly (5% level) speeded up by all other treatments (Table 1) . Effect of pathogenic and non-pathogenic fungi and fungal extracts on abscission in

G . barbadense Cotyledons of the cv . Pima were inoculated by applying 19 . 6 cm2 filter paper discs dipped in an inoculum of about 9 x 10 3 spores ml - ' of A . macrospora, or A . alternata (pathogens) or about 20 x 10-3 spores ml -' of Fusarium oxysporum f. sp . lycopersici or Botrytis cinerea (non pathogens) . Other treatments included injecting spore suspensions



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TABLE 2

The effect of inoculation with pathogenic and non-pathogenic fungi or treatment with cell-free ficngal extracts on the abscission of cotyledons by G . barbadense

Area affected by Treatment

Untreated Inoculated with' : A . macrospora A . alternata F. oxysporum B . cinerea Injected with spore suspension of : A . macrospora A . alternata F. oxysporum B . cinerea Injected with cell-free extract of : A . macrospora .4 . alternata F. oxysporum B . cinerea

Necrosis

Disease

Shedding

45 + 2° 73 19

17+ 1 35+2 45+1 39+2

60 30 1 .4 21

18+1 34+1 37+3 38+2

60 6 68 47

32± I 26+2 35+2 33+3

0.3 0.3

'Days after treatments applied . ° Inoculum concentrations of A . macrospora, and A . alternata .

of each fungus, or their cell-free extacts, into cotyledons . Table 2 shows that A . alternata infections produced less severe disease than A . macrospora while inoculations with F. oxysporum and B . cinera as expected caused practically no disease . Relative to controls, abscission of cotyledons was most rapid when the seedlings were infected with A . macrospora, was only slightly speeded up by infection with A . alternata, and was not, or little affected by inoculation with F. oxysporum and B . cinerea . Injection of live spores or of cell-free extracts of the four fungi induced either disease with the pathogen being present in the lesions or necrosis without the pathogen being present in the lesion . Injection of live spores and cell free extracts speeded up shedding but the rate was not always proportional to the area of the damaged tissue . For instance, spores or extracts of B . cinerea caused more necrosis than those of F . oxysporum, but both fungi had a similar effect on shedding . Live spores of A . macrospora affected shedding more than those of A . alternata, but cell-free extracts of A . alternata had a strong effect on shedding despite relatively little necrosis . DISCUSSION In G . barbadense the rate of abscission of cotyledons infected with A . macrospora was directly related to the severity of the disease, was higher in plants grown at high preinoculation temperatures than at low pre-inoculation temperatures ; and was higher in plants grown with low levels of host nutrition than in well fertilized plants . Abscission of infected cotyledons of G . hirsutum was also induced by infection but it occurred later



299 Cotyledons of G . barbadense and G . hirsutum probably exemplifies a than in G . barbadense . The delay in the response of G . hirsutum host effect not necessarily connected with the development of disease . Differential effects on leaf abscission are known from other host-parasite systems . For example, infection with Leveillula taurica, induced premature shedding in peppers but not in tomatoes [8] . Also, some cultivars of peanut shed their leaves more rapidly than others in response to infection by Cercospora arachidicola [4] . The present study showed that defoliation can be induced by infection, by various chemical treatments, mechanical injury and by incubation in continuous darkness . The whole range of agents, biotic, chemical and physical, that induce abscission in cotton, suggests that abscission is a non specific response to stress . It is not known whether the action of stress agents is associated with the development of a unique senescence system or results from acceleration of a more common system, like inactivation of auxins that tend to reduce shedding [10] or increased production of ethylene that promotes abscission [4] . In nature, premature defoliation in the sensitive cotton cultivars is exerted mainly by the action of .4 . macrospora because this, rather than other agents of stress, happens to be present in the field .

REFERENCES F . T . (1982) . Abscission . 369 pp. University of California Press . E ., ROTEM, J ., PINNSCHMIDT, H . & KRANZ, J . (1983) . Influence of controlled environment and age on development of Alternaria macrospora and on shedding of leaves in cotton. Phytopathology 73, 1145-1147 . 3 . BASHI, E ., SACHS, Y . & ROTEM, J . (1983) . Relationships between disease and yield in cotton fields affected by Alternaria macrospora . Phytoparasitica 11, 89-97 . 4 . KETRING, D . L . & MELOUK, H . A . (1982) . Ethylene production and leaflet abscission in three peanut genotypes infected with Cercospora arachidicola Hori . Plant Physiology 69, 789-792 . 5 . KRANZ, J . (1976) . Einfluss einiger Pflanzenkrankheiten auf den Abgang der Blätter . Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 83, 234-237 . 6 . OSBORNE, D . J . (1968) . Hormonal mechanisms regulating senescence and abscission . In . Biochemistry and Physiology of Plant Growth Substances . Ed . by F . Wightman and G . Setterfield, pp . 815-840 . Runge Press, Ottawa. 7 . REUVENI, R ., PERL, M . & ROTEM, J. (1974) . The effect of Leveillula taurica on leaf abscission in peppers . Phytopathologische Zeitschrift 80, 79-84 . 8 . REUVENI, R . & ROTEM, J . (1973) . Epidemics of Leveillula taurica on tomatoes and peppers as affected by the conditions of humidity . Phytopathologische Zeitschrift 76, 153-157 . 9 . SCHEIN, R . D . (1964) . Design, performance, and use of a quantitative inoculatpr . Phvtopathology 54, 509-513 . 10 . SEQUEIRA, L. & STEEVES, T . A . (1954) . Auxin inactivation and its relation to leaf drop caused by the fungus Omphalia flavida . Plant Physiology 29, 11-16 . 11 . SEXTON, R . & ROBERTS, J . A. (1982) . Cell biology of abscission . Annual Review of Plant Physiology 33, 133-162 . 12 . WIESE, M . V . & DEVAY, J . E. (1970) . Growth regulator changes in cotton associated with defoliation caused by Verticillium albo-atrum . Plant Physiology 45, 304-309 . 13 . WILLIAMSON, C . E . (1950) . Ethylene, a metabolic product of diseased or injured plants. Phytopathologv 40, 205-208 . 14 . YARWOOD, C . E . (1967) . Response to parasites . Annual Review of Plant Physiology 18, 419-438 . 1 . ADDICOTT,

2.

BASHI,