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Sensitivity to gamma irradiation of fungi pathogenic to pears
International JoumalofAppliedRadiation and 1sotopca,1969,Vol. 2O,pp.577-585.Pcrgamon Pms. Printed in NorthernIreland
Sensitivity to Gamma Irradiation of Fungi Pathogenic to Pears* RIVKA BARKAI-GOLAN, RUTH BEN-ARIE and SYLVIA
GUELFAT The Volcani Institute of Agricultural Research, Bet Dagar, Israel
REICH
and R. S. KAHAN Soreq Nuclear Research Centre, Yavne, Israel (Received 15 February 1969) The effect of gamma irradiation at doses of 50-300 krad, on the germination ability of fimgi in vitro, and prolongation of the incubation period in vivo, was studied with known spore populations of four fungi pathogenic to stored pears. Rot development and fungal control were examined in cold storage after irradiation of fruit inoculated with two radio-sensitive fungi. Inhibition of spore germination was found to be directly correlated to the strength of the radiation dose and indirectly correlated to the spore concentration. Penicilkum e.+nsum was the most sensitive to radiation both in culture and in fruit tissues. Some prolongation of the incubation period of P. e+nsum and of Botrytis cinerea, the two principal pathogens, occurred after irradiation with 100 krad. With increasing dosage, the incubation period was more prolonged and staggered and the percentage of infected fruit began to decrease from 200 krad. Altemaria tenuis and Stemjhylium botryosum were radiation-resistant. In spite of a decrease in germination ability at the higher doses, the pathogenicity and incubation period of both these fungi were not significantly affected. LA SENSIBILITE
AU RAYONNEMENT
PATHOGENIQUES
AUX
GAMMA
DES FONGUS
POIRES
On a ttudiC I’effet du rayonnement gamma a des doses de 50-300 krad sur la capabilitt germinative des fongus in vitro, et la prolongation de la p&ode d’incubation in vim, avec des populations de spores connues de quatre fungus pathogtiques aux poires en magasin. On regarda le d&eloppement de la pourriture et le contr6le des fongus dam la chambre froide suivant l’irradiation des fruits qu’on avait inocul& de deux fongus radio-sensibles. On trouva l’inhibition de la germination des spores en rapport direct h la puissance de la dose de rayonnement et en rapport indiract a la concentration des spores. Le Ptiillium Ex@nsum fut le plus sensible au rayonnement et en culture et dans les tissus des fruits. 11y eut un prolongement quelconque de la pCriode d’incubation du P. Ex@u-um et du Botrytis Cinkrka, les duex principaux pathog*nes, suivant une irradiation de 100 krad. Avec augmentation de la dose, la p&ode d’incubation devint plus prolong&e et deplacte et la proportion de fruit infactt vint & se diminuer a partir de 200 krad. L’Alternatia Tkruis et le Stemphylium Botryosum furent rCsistantsau rayonnement. Malgrt une diminution de la capabilitb germinative aux doses plus fortes, il n’y eut pas d’effet signifiant sur la pathogt+nicitCou sur la p&ode d’incubation de ces deux fongus. gYBCTBMTEJIbHOCTb
IIATOrEHHbIX JJJIH I’PYIJI l?PBBKOB H3JIY=IEHEIIO
K I’AMMA
Hsvanocb Ae&cTBEe06JIy¶eHHRranaana-sysamnB noaax OT 60 no 300 ~pm Ha cnoco6aoc~1, KpOpaCTaAHHFPH6KOBi~evitro(B~a6opaTopHbnrOK~~),aTaKHceHay~~HeHEeH~Ky6~~0HHOrO IIepHO~aintiWO (B )f(EBblXOPl'~HTMPX): eKC~e~HMeHTbl~OH6BO~HnHCbnpEnO~~qSiIiCIIOpElMK ¶eTblpeX BKAOB FPH6HOB, UaTOl'eHHblX AJIH rpyIII BO BpeMH XpaIieHEH. PasBHTAe BarHFfBaHHH IIpOBepHJIOCb Ii KOHTpOJIb rpK6Ko~ npOE~BO~JICH npa XOJIOAHOM xpaH~HHHII~~JI~~~JI~~~AKH(P~~KToB, 8apameHnbnrAByrriBA&~MK¶~BCTBHT~JI~R~IXK~~JQ'~~H~C rpSi6KOB. * Contribution from The Volcani Institute of Agricultural Research, Bet Dagan, Israel. No. 1413-E. 577
1968 Series,
578
Rivka Barkai-Golan, Ruth Ben-Arie, Sylvia Guelfat-Reich and R. S. Kahan OGKapymnnocb, ~To swepxKa npopacTaHHHcnop HaxoAnTcHB npamom cooTHomenHHc MOIUHOCTbIo ~osn o6nyqemrr II B H0CBeHHoM-c HoHqeHTpaqHet cnop. HaKBonee SYBCTBKTenbnm K o6nyqeKKm KaK B KynbType, TaK H B TKanKKX @PYKTOB oKasancR: rpa60~
Penicillium expanaum. HeKoTopoe yHnaHeHmeHHHy6aqHoHHoronepaona H~yx rnaBHbIX naToreHHbIx BHAOB, Penicilliunr expnau?n H Botrytie cinweq aa6nmnaaocb nocne 06ny9eHnK ~oaoti B 100 spa& C n0BwneKKer.i gO8bI HHHy6aHHoHHnfinepaog Hone6ancs H sy~nu~HncH elqe 6onee, a npu Hose B 200 HpaH npoqeHT sapaHteHH= @PYKTOBna¶HHan yMeHbU.laTbCFl. BHAU Alternaricc ten&e H Stemphyliurn botryosum oKaaanHcb YCTO~~UHBJIIMH K o6nyHeHHro. XOTR.HeKoTopoenOHnmenuecnoco6HocTH npopacTaHHHnpri 6onee B~ICOKEIX AOaaXHa6HIOHanocb,HOHaTOreHHOCTb H HHHy6aHHOHHhlB nepUOgBTHX HByX BEiAOB rpa6KoB He &GlMeHflJlMCbB CKOnbKO-HU6yab
EMPFINDLICHKEIT
!%a=iHTeJlbHOltf Mepe.
VON FUR BIRNEN PATHOGENISCHEN UBER GAMMABESTRAHLUNG
PILZEN
GEGEN-
Der Einfluss von Gammabestrahlung mit Dosen von 50-300 krad aufdie Keimfahigkeit von Pilzen in vitro und die Verl&ngerung der Brutzeit in vivo wurde mit bekannten Besetzungsdichten von vier fiir gelagerte Birnen pathogen&he Pilze untenucht. Faulnisentwicklung und Pilzbekiimpfung wurden in einem Ktihlhaus nach Bestrahlung von mit zwei strahlenempfindlichen Pilzen geimpften Frtichten geprtift. Die Hemmung der Pilzkeimung fand man ah in direkter Beziehung zu der StLke der Strahlungsdose und indirekt zu der Sporenkonzentration stehend. Penicillium ex@nsum war am empfindlichsten fur Strahlung sowohl in Kultur als such im Fruchtgewebe. Eine gewisse Verlfingerung der Brutzeit von P. [email protected] und von Botrytis cinerea, den zwei Hauptpathogenen, trat nach Bestrahhmg mit 100 krad auf. Bei steigenden Dosen wurde die Brutzeit liinger und schwa&e und der Anteil von infizierten Frtichten begann von 200 krad an zu fallen. Altemaria tenuis und Stemjhylium botryosum waren strahlungsfest. Trotz der verminderten Keimf%igkeit mit den hoheren Dosen waren die pathogenische Wirkung und Brutzeit beider Pilze nicht merklich beeinflusst.
INTRODUCTION THE SENSITIVITY of rot-causing fungi to gamma irradiation has been described with regard to fungi pathogenic to citrus fruits,(l-s) plum~,(~~7) strawberries,t7**) grapes,(s*B-ll) and melons(12). In the present work the direct effects of gamma irradiation on the in vitro germination and the in vivo development of fungi pathogenic to stored pears were examined as part of a comprehensive study of the effects of gamma irradiation on the storage life of pears. The principal species which cause fimgal rots of stored Spadona pears in Israel are Penicillium expansum Link., Botrytis cinerea Pers., Alternaria tenuis Nees and Stemfihylium botryosum Wall. The first two fungi, which cause the majority of storage rots and are relatively resistant to the chemical control means already knowno*), are amongst those fungi which respond to relatively low doses of gamma irradiation. The dose response curves drawn by SOMMER et al.‘@ for Penicillium expansum and Botrytis cinerea isolated from Prunus fruits show a considerable decrease in the percentage
of germination
as a result of
irradiation with doses of 50 and 150 krad, respectively. Irradiation of inoculum of B. cinerea, containing both mycelium and spores, with a dose of 200 krad, resulted in a marked prolongation of the incubation period of the fungus in inoculated strawberries’s). To the best of our knowledge, the pathogenicity of the above fungi following both in vitro irradiation and irradiation within the inoculated pear has not yet been studied. MATERIALS
AND METHODS
Spore suspensions of the four above-mentioned fungi, prepared at concentrations of 1Os, 1O3and 1O4 spores/ml from pure 14-18 day old culture grown on PDA, were irradiated with doses of 50, 100,200 and 300 krad of 6oCo gamma rays in a Gammacell 200 at a rate of 360 krad/hr. An aliquot of O-25 ml of irradiated suspension, diluted according to the estimated percentage of spore germination, was plated on PDA in a petri dish. The developing colonies were counted microscopically after 24 hr at 23”C, and again, macroscopically, after three and five
579
Sensitivity to gamma irradiationoffungi pathogenicto pears days, in order to designate colonies which showed retarded development. Each treatment consisted of 12 replicates. Examination of the pathogenicity of the irradiated fungi was carried out by inoculating pear fruits (Pyrus communis L. var. Spadona) with irradiated suspensions. Aliquots of 0.05 ml from suspensions containing 10s spores/ml which had been irradiated with doses of 50-300 krad, were inserted through a window-like wound to a depth of 3 mm, on the cheek of the fruit. After inoculation, the wound was sealed with cellotape and the fruit was held at room temperature for 24 days. The incubation period of the fungi in the fruit was examined daily. Examination of the pathogenicity of the fungi after in viva irradiation was carried out by irradiating previously inoculated pears. The fruit was inoculated in the manner described above on the day after harvest with spore suspensions of P. expattsum and B. cineria containing lo4 spores/ml. The volume of the inoculum in this experiment was 0.02 ml. The inoculated fruit was held at room temperature for 18 hr before irradiation with doses of 50, 100,200 and 300 krad. After treatment, the fruit was held under simulated storage conditions:-paper wrapped, packed in polyethylene liners and
stored at 0°C. The fruit was examined daily during the first fortnight, and from then on twice a week, for those fruits that still showed no signs of rot. The growth rate of the rot was measured once a week. At the end of the storage period the fruit was held for one week at room temperature to ascertain whether the nondecayed fruit remained healthy during shelf-life. Each of the in vivo treatments consisted of 40 inoculations, two per fruit. RESULTS In vitro tests Inhibition of spore germination was found to be in direct proportion to the strength of the dose and in inverse proportion to the concentration of spores in the irradiated suspension. The effect of irradiation doses on the percentage of spore germination on PDA at 23°C is shown in Table 1. Significant differences between the irradiation doses and the spore concentrations were found for P. expansum, B. cinerea and S. botryosum (p = O-01). For A. tenuis only the 300 krad dose differed significantly from the control. Penicillium expansum was the fungus most susceptible to irradiation: the lOO-krad dose caused in vitro inactivation of all the spores at concentrations of 1Oa and 10s spores/ml. A
TABLE 1. The effect of gamma irradiation on the germination ability in vitro
of pear pathogens. (percent germination on PDA, control being consideredas 100%)
Pathogen Penidlium exfiatwm
Botrytis cinerea
Stemjhylium Botryosum
Alternaria teat&
Dose (krad) 50
100 200 300 50
100 200 300 50 100 200 300 50
100 200 300
Population density (spores/ml) 10s
10s
104
25 0 0 0 45 18 0 0 60 45 38 15
45 0 0 0 70 56 o-2 0 75 53 40 35
60 0.4 0 0 85 70 o-5 0.02 90 80 57 40 100 100
100 90 92
100 90 100
100
58
70
70
Rivka Barkai-Golan,Ruth Ben-Arie, Sylvia Guelfat-Reichand R. S. Kahan
580
200~krad dose resulted in total inactivation of B. cinerea spores at the lowest spore concentration (lOa/ml). A. tenuis and S. botryosum were resistant to all the doses examined, and even at 300 krad the percentage of germination was high. Although the 50-krad dose reduced the germination rate of S. botryosum, further marked reduction was not obtained by increasing the dosage up to 200 krad. A noticeable reduction in the germination rate ofA. tenuis began to be apparent only at 300 krad. In vivo tests (a) Pathogenicity of irradiated fungal sus@nsions. Inoculation of the fruit with irradiated spore suspensions also showed the greater susceptibility of P. expansum to gamma irradiation (Fig. 1).
F=-a. Rniciltiim apansum
10° 60
b. Botrytis dnema
Jr
I
! f ! i
60 -40
The 50-krad dose already resulted in a three day prolongation of the incubation period of the fungus in the majority of inoculated fruits (85 per cent) in comparison with the control. A more noticeable prolongation of the incubation period to 17 days in those fruit which rotted, was obtained by the 100~krad treatment. In the majority of fruits (66 per cent) there was no rot development whatsoever by the end of the 1&day storage period. A certain prolongation of the incubation period of B. cinerea in the fruit occurred after irradiation of the spore suspension with 100 krad. Whereas in the control fruit the length of the incubation period was uniform in all the inoculated fruits, in the irradiated fruits there was wide variability in the incubation periods. In the latter, the first signs of rot appeared within four days in a number offruits (8 per cent)
Stemphylium botryosum
0
4
8
12
d. AhffMrla
16 DAYS~TER?tN&“LAhON
tenuis
8121620X
-0 kmd 0 . . . ..-.. 0 CjOk& o----al00 o-.-.-o ~300
kmd 200 kmd kmd
Fxo. 1. The incubation period and percentage of rot in Spadona pears inoculated with irradiated spore suspensions of four fungi (fruit held at room temperature).
Sensitivity to gamma irradiationofjhgi pathogenicto pears
tion of the incubation period of P. expansum and B. cinerea with increased dosage (Figs. 2 and 3).
but incubation was prolonged up to 24 days in the remaining fruit. Although irradiation of spore suspensions of A. tenuis and S. botryosum resulted in some inhibition of the germination ability of the spores in vitro (Table l), it did not prevent rot development in the inoculated fruits (Fig. 1). However, a noticeable prolongation of the incubation period of S. bob-yosum in the fruit occurred with 200 krads-8-19 days vs. the 4-11 days for control fruit. The incubation period of A. tenuk was not significantly lengthened as a result of irradiation. The slight increase which occurred at the lower doses was not enhanced by increasing the dosage. (b) Irradiation of inoculated &it. Irradiation of inoculated fruits showed a gradual prolonga,
,
,
,
a. Penicillium
,
,
,
As with fruit inoculated with irradiated suspensions, the marked result of irradiation, which increased with the dose, was the staggering effect on the incubation period. However, once the rot had begun to develop, its growth rate was more or less constant (Fig. 2). Inhibition of pathogenicity was noticeable from 200 krad, but it was much more marked with P. expsum than with B. cinerea (Fig. 3). Although a noticeable prolongation of an average of ten days in the incubation period of P. expanwm was obtained with the 100~krad dose, noticeable rot control occurred only at 200 krad. With this dose, 2.5 per cent of the fruit began to rot within 17 days and rot control of 60 per ,
expansum
,
,
,
,
,
,
,
P
b. Batrytis cinerea P
-
0 krad
o----O
50
o----o100 *--q-3
581
krad kmd
200 kmd
-3COkrad
FIG. 2. The effect of gamma irradiation on the growth rate of rots of Spadona pears stored at 0%.
582
Rivka Barkai-Golan,Ruth Ben-Arie, Sylvia Gue~at-Reichand R. S. Kahan ,
Cl?,, ; ,’ : :
,
(,
0. Peniciltium
,
,
,
,
,
,
expansum
8 : ,I ;
:: s !f
-
<
0
b. Botrytis cinerea
0
6
16
W
0 krad
O-..-----0
50 kmd
O----0100
24 M’S
32 40 46 AFTER INOCi ILATlON
56
64
72
kmd
o-.-.-O
200 kmd
o----O
300 kmd
FIG. 3. The effect of gamma irradiation on the length of the incubation period and the percentage of rot development in inoculated Spadona pears stored at 0%. cent was achieved within two months’ storage. On the other hand, 50 per cent of the fruit inoculated with B. cinerea and irradiated with 300 krad, showed signs of rot within 33 days of inoculation.
DISCUSSION
AND CONCLUSIONS
P. expansum spores
in a suspension of 10s spores/ml entirely lost their germinating ability on a PDA medium following gamma irradiation with a lOO-krad dose, whereas they retained their ability to cause fruit rot. Also in fruit inoculated with a 104/ml spore suspension of P. expansum, the fungus developed after irradiation with 100 krad and some fruit was infected even following irradiation with 200 and 300 krad. However, it is important to note that, at
the time of irradiation, the fungus had already begun to invade the fruit tissues and irradiation therefore did not affect the freshly collected spores, but rather spores at various stages of germination. It was similarly found that, whereas a dose of 200 krad inactivated 99.8 per cent of B. cinerea spores in vitro in a suspension of lo3 spores/ml, all the fruit inoculated with the same suspension was infected. These results are in agreement with the higher lethal dose of gamma rays described by BERAHA~~) for mycelium of B. cinerea within the host, in comparison with artificial culture media; and also with the total inhibition of colonial growth of Botrytis in culture obtained by a 300-krad dose as compared to the ineffectiveness of the same dose in controlling
Sensitivity to gamma iwadziation offungi pathogenic to @us the pathogenicity of the fungus to strawberries under the same conditions(s). The in vivo experiments show that irradiation with gamma rays resulted in a noticeable prolongation of the incubation periods of both P. exparuum and B. cinerea-tie two most common causal agents of storage rots in Spadona pears. Even though partial control of both fungi was obtained only at relatively high doses, in nature the actual concentration of spores with which the fruit is in contact at harvest is much lower, and the effect of irradiation is likely to be more marked.@*“*s) On the other hand, doses exceeding 200 krad have been found to prevent normal ripening of this fruitos).
REFERENCES 1. BERAHA L., RAMSEY G. B., SMITH M. A. and WRIGHT W. R. Phytofiathology 49, 91 (1959). 2. BARKA&~L.AN R. and KAHAN R. S. Prelim. Refit. Nat. Univ. Inst. Agr. Rehovot, Israel, No. 511
(1965) (in Hebrew, with English summary). 3. SOMMERN. P., MAXIE E. C., FORTLAGER. J. and ECKERTJ. W. Rad. Bot. 4, 317 (1964).
583
4. BARKAI-GOLAN R. and KAHANR. S. Plant Dis. Rep. 50,874 (1966). 5. SARAVACOS G. D., HATZIPETROU L. P. and GEOROIADOUE. Food Zwad. 3, A6 ( 1962). 6. SOMMERN. P., MAXIE E. C. and FORTLAGER. J. Rad. Bot. 4,309 (1964). 7. SOPOLOV G. P., Radiobiologiya 4,460 (1964). 8. BARKAI-GOLAN R., TEMKIN-G~RODEISKIN. and KAHAN R. S. Food Iwad. 8, 34 (1967). 9. NELSON K. E., MAXIB E. C. and E~KEL W. Phytofiathology 49,475 (1959). 10. COUEY H. M. and BRAMLAGE W. J. Phytopathology 558, 1013 (1965). 11. BARKAI-GOLAN R. and KAHAN R. S. Prelim. Rep. Nat. Univ. Agr. Rehovot. Israel, No. 605 (1968) (in Hebrew, with English summary).
12. BARKAI-GOLAN R., KAHAN R. S. and TEMKINGORODEISKIN. 579 (1968).
13. BEN-AFUE R.
Int. J. a#.
Radiat.
Isotopes 19,
Alon Hanotea 25 (4), 189 (1968)
(in Hebrew).
14. BERAHA L., SMITH M. A. and WRIGHT W. R. Phytojathology 58,474 ( 1960). 15. GUELFAT-REICH S., BEN-ARIE R., KAHAN R. S. and EISENBERGE. J. Hart. Sci. 44 (2), (1969).
Sensitivity to Gamma Irradiation of Fungi Pathogenic to Pears* RIVKA BARKAI-GOLAN, RUTH BEN-ARIE and SYLVIA
GUELFAT The Volcani Institute of Agricultural Research, Bet Dagar, Israel
REICH
and R. S. KAHAN Soreq Nuclear Research Centre, Yavne, Israel (Received 15 February 1969) The effect of gamma irradiation at doses of 50-300 krad, on the germination ability of fimgi in vitro, and prolongation of the incubation period in vivo, was studied with known spore populations of four fungi pathogenic to stored pears. Rot development and fungal control were examined in cold storage after irradiation of fruit inoculated with two radio-sensitive fungi. Inhibition of spore germination was found to be directly correlated to the strength of the radiation dose and indirectly correlated to the spore concentration. Penicilkum e.+nsum was the most sensitive to radiation both in culture and in fruit tissues. Some prolongation of the incubation period of P. e+nsum and of Botrytis cinerea, the two principal pathogens, occurred after irradiation with 100 krad. With increasing dosage, the incubation period was more prolonged and staggered and the percentage of infected fruit began to decrease from 200 krad. Altemaria tenuis and Stemjhylium botryosum were radiation-resistant. In spite of a decrease in germination ability at the higher doses, the pathogenicity and incubation period of both these fungi were not significantly affected. LA SENSIBILITE
AU RAYONNEMENT
PATHOGENIQUES
AUX
GAMMA
DES FONGUS
POIRES
On a ttudiC I’effet du rayonnement gamma a des doses de 50-300 krad sur la capabilitt germinative des fongus in vitro, et la prolongation de la p&ode d’incubation in vim, avec des populations de spores connues de quatre fungus pathogtiques aux poires en magasin. On regarda le d&eloppement de la pourriture et le contr6le des fongus dam la chambre froide suivant l’irradiation des fruits qu’on avait inocul& de deux fongus radio-sensibles. On trouva l’inhibition de la germination des spores en rapport direct h la puissance de la dose de rayonnement et en rapport indiract a la concentration des spores. Le Ptiillium Ex@nsum fut le plus sensible au rayonnement et en culture et dans les tissus des fruits. 11y eut un prolongement quelconque de la pCriode d’incubation du P. Ex@u-um et du Botrytis Cinkrka, les duex principaux pathog*nes, suivant une irradiation de 100 krad. Avec augmentation de la dose, la p&ode d’incubation devint plus prolong&e et deplacte et la proportion de fruit infactt vint & se diminuer a partir de 200 krad. L’Alternatia Tkruis et le Stemphylium Botryosum furent rCsistantsau rayonnement. Malgrt une diminution de la capabilitb germinative aux doses plus fortes, il n’y eut pas d’effet signifiant sur la pathogt+nicitCou sur la p&ode d’incubation de ces deux fongus. gYBCTBMTEJIbHOCTb
IIATOrEHHbIX JJJIH I’PYIJI l?PBBKOB H3JIY=IEHEIIO
K I’AMMA
Hsvanocb Ae&cTBEe06JIy¶eHHRranaana-sysamnB noaax OT 60 no 300 ~pm Ha cnoco6aoc~1, KpOpaCTaAHHFPH6KOBi~evitro(B~a6opaTopHbnrOK~~),aTaKHceHay~~HeHEeH~Ky6~~0HHOrO IIepHO~aintiWO (B )f(EBblXOPl'~HTMPX): eKC~e~HMeHTbl~OH6BO~HnHCbnpEnO~~qSiIiCIIOpElMK ¶eTblpeX BKAOB FPH6HOB, UaTOl'eHHblX AJIH rpyIII BO BpeMH XpaIieHEH. PasBHTAe BarHFfBaHHH IIpOBepHJIOCb Ii KOHTpOJIb rpK6Ko~ npOE~BO~JICH npa XOJIOAHOM xpaH~HHHII~~JI~~~JI~~~AKH(P~~KToB, 8apameHnbnrAByrriBA&~MK¶~BCTBHT~JI~R~IXK~~JQ'~~H~C rpSi6KOB. * Contribution from The Volcani Institute of Agricultural Research, Bet Dagan, Israel. No. 1413-E. 577
1968 Series,
578
Rivka Barkai-Golan, Ruth Ben-Arie, Sylvia Guelfat-Reich and R. S. Kahan OGKapymnnocb, ~To swepxKa npopacTaHHHcnop HaxoAnTcHB npamom cooTHomenHHc MOIUHOCTbIo ~osn o6nyqemrr II B H0CBeHHoM-c HoHqeHTpaqHet cnop. HaKBonee SYBCTBKTenbnm K o6nyqeKKm KaK B KynbType, TaK H B TKanKKX @PYKTOB oKasancR: rpa60~
Penicillium expanaum. HeKoTopoe yHnaHeHmeHHHy6aqHoHHoronepaona H~yx rnaBHbIX naToreHHbIx BHAOB, Penicilliunr expnau?n H Botrytie cinweq aa6nmnaaocb nocne 06ny9eHnK ~oaoti B 100 spa& C n0BwneKKer.i gO8bI HHHy6aHHoHHnfinepaog Hone6ancs H sy~nu~HncH elqe 6onee, a npu Hose B 200 HpaH npoqeHT sapaHteHH= @PYKTOBna¶HHan yMeHbU.laTbCFl. BHAU Alternaricc ten&e H Stemphyliurn botryosum oKaaanHcb YCTO~~UHBJIIMH K o6nyHeHHro. XOTR.HeKoTopoenOHnmenuecnoco6HocTH npopacTaHHHnpri 6onee B~ICOKEIX AOaaXHa6HIOHanocb,HOHaTOreHHOCTb H HHHy6aHHOHHhlB nepUOgBTHX HByX BEiAOB rpa6KoB He &GlMeHflJlMCbB CKOnbKO-HU6yab
EMPFINDLICHKEIT
!%a=iHTeJlbHOltf Mepe.
VON FUR BIRNEN PATHOGENISCHEN UBER GAMMABESTRAHLUNG
PILZEN
GEGEN-
Der Einfluss von Gammabestrahlung mit Dosen von 50-300 krad aufdie Keimfahigkeit von Pilzen in vitro und die Verl&ngerung der Brutzeit in vivo wurde mit bekannten Besetzungsdichten von vier fiir gelagerte Birnen pathogen&he Pilze untenucht. Faulnisentwicklung und Pilzbekiimpfung wurden in einem Ktihlhaus nach Bestrahlung von mit zwei strahlenempfindlichen Pilzen geimpften Frtichten geprtift. Die Hemmung der Pilzkeimung fand man ah in direkter Beziehung zu der StLke der Strahlungsdose und indirekt zu der Sporenkonzentration stehend. Penicillium ex@nsum war am empfindlichsten fur Strahlung sowohl in Kultur als such im Fruchtgewebe. Eine gewisse Verlfingerung der Brutzeit von P. [email protected] und von Botrytis cinerea, den zwei Hauptpathogenen, trat nach Bestrahhmg mit 100 krad auf. Bei steigenden Dosen wurde die Brutzeit liinger und schwa&e und der Anteil von infizierten Frtichten begann von 200 krad an zu fallen. Altemaria tenuis und Stemjhylium botryosum waren strahlungsfest. Trotz der verminderten Keimf%igkeit mit den hoheren Dosen waren die pathogenische Wirkung und Brutzeit beider Pilze nicht merklich beeinflusst.
INTRODUCTION THE SENSITIVITY of rot-causing fungi to gamma irradiation has been described with regard to fungi pathogenic to citrus fruits,(l-s) plum~,(~~7) strawberries,t7**) grapes,(s*B-ll) and melons(12). In the present work the direct effects of gamma irradiation on the in vitro germination and the in vivo development of fungi pathogenic to stored pears were examined as part of a comprehensive study of the effects of gamma irradiation on the storage life of pears. The principal species which cause fimgal rots of stored Spadona pears in Israel are Penicillium expansum Link., Botrytis cinerea Pers., Alternaria tenuis Nees and Stemfihylium botryosum Wall. The first two fungi, which cause the majority of storage rots and are relatively resistant to the chemical control means already knowno*), are amongst those fungi which respond to relatively low doses of gamma irradiation. The dose response curves drawn by SOMMER et al.‘@ for Penicillium expansum and Botrytis cinerea isolated from Prunus fruits show a considerable decrease in the percentage
of germination
as a result of
irradiation with doses of 50 and 150 krad, respectively. Irradiation of inoculum of B. cinerea, containing both mycelium and spores, with a dose of 200 krad, resulted in a marked prolongation of the incubation period of the fungus in inoculated strawberries’s). To the best of our knowledge, the pathogenicity of the above fungi following both in vitro irradiation and irradiation within the inoculated pear has not yet been studied. MATERIALS
AND METHODS
Spore suspensions of the four above-mentioned fungi, prepared at concentrations of 1Os, 1O3and 1O4 spores/ml from pure 14-18 day old culture grown on PDA, were irradiated with doses of 50, 100,200 and 300 krad of 6oCo gamma rays in a Gammacell 200 at a rate of 360 krad/hr. An aliquot of O-25 ml of irradiated suspension, diluted according to the estimated percentage of spore germination, was plated on PDA in a petri dish. The developing colonies were counted microscopically after 24 hr at 23”C, and again, macroscopically, after three and five
579
Sensitivity to gamma irradiationoffungi pathogenicto pears days, in order to designate colonies which showed retarded development. Each treatment consisted of 12 replicates. Examination of the pathogenicity of the irradiated fungi was carried out by inoculating pear fruits (Pyrus communis L. var. Spadona) with irradiated suspensions. Aliquots of 0.05 ml from suspensions containing 10s spores/ml which had been irradiated with doses of 50-300 krad, were inserted through a window-like wound to a depth of 3 mm, on the cheek of the fruit. After inoculation, the wound was sealed with cellotape and the fruit was held at room temperature for 24 days. The incubation period of the fungi in the fruit was examined daily. Examination of the pathogenicity of the fungi after in viva irradiation was carried out by irradiating previously inoculated pears. The fruit was inoculated in the manner described above on the day after harvest with spore suspensions of P. expattsum and B. cineria containing lo4 spores/ml. The volume of the inoculum in this experiment was 0.02 ml. The inoculated fruit was held at room temperature for 18 hr before irradiation with doses of 50, 100,200 and 300 krad. After treatment, the fruit was held under simulated storage conditions:-paper wrapped, packed in polyethylene liners and
stored at 0°C. The fruit was examined daily during the first fortnight, and from then on twice a week, for those fruits that still showed no signs of rot. The growth rate of the rot was measured once a week. At the end of the storage period the fruit was held for one week at room temperature to ascertain whether the nondecayed fruit remained healthy during shelf-life. Each of the in vivo treatments consisted of 40 inoculations, two per fruit. RESULTS In vitro tests Inhibition of spore germination was found to be in direct proportion to the strength of the dose and in inverse proportion to the concentration of spores in the irradiated suspension. The effect of irradiation doses on the percentage of spore germination on PDA at 23°C is shown in Table 1. Significant differences between the irradiation doses and the spore concentrations were found for P. expansum, B. cinerea and S. botryosum (p = O-01). For A. tenuis only the 300 krad dose differed significantly from the control. Penicillium expansum was the fungus most susceptible to irradiation: the lOO-krad dose caused in vitro inactivation of all the spores at concentrations of 1Oa and 10s spores/ml. A
TABLE 1. The effect of gamma irradiation on the germination ability in vitro
of pear pathogens. (percent germination on PDA, control being consideredas 100%)
Pathogen Penidlium exfiatwm
Botrytis cinerea
Stemjhylium Botryosum
Alternaria teat&
Dose (krad) 50
100 200 300 50
100 200 300 50 100 200 300 50
100 200 300
Population density (spores/ml) 10s
10s
104
25 0 0 0 45 18 0 0 60 45 38 15
45 0 0 0 70 56 o-2 0 75 53 40 35
60 0.4 0 0 85 70 o-5 0.02 90 80 57 40 100 100
100 90 92
100 90 100
100
58
70
70
Rivka Barkai-Golan,Ruth Ben-Arie, Sylvia Guelfat-Reichand R. S. Kahan
580
200~krad dose resulted in total inactivation of B. cinerea spores at the lowest spore concentration (lOa/ml). A. tenuis and S. botryosum were resistant to all the doses examined, and even at 300 krad the percentage of germination was high. Although the 50-krad dose reduced the germination rate of S. botryosum, further marked reduction was not obtained by increasing the dosage up to 200 krad. A noticeable reduction in the germination rate ofA. tenuis began to be apparent only at 300 krad. In vivo tests (a) Pathogenicity of irradiated fungal sus@nsions. Inoculation of the fruit with irradiated spore suspensions also showed the greater susceptibility of P. expansum to gamma irradiation (Fig. 1).
F=-a. Rniciltiim apansum
10° 60
b. Botrytis dnema
Jr
I
! f ! i
60 -40
The 50-krad dose already resulted in a three day prolongation of the incubation period of the fungus in the majority of inoculated fruits (85 per cent) in comparison with the control. A more noticeable prolongation of the incubation period to 17 days in those fruit which rotted, was obtained by the 100~krad treatment. In the majority of fruits (66 per cent) there was no rot development whatsoever by the end of the 1&day storage period. A certain prolongation of the incubation period of B. cinerea in the fruit occurred after irradiation of the spore suspension with 100 krad. Whereas in the control fruit the length of the incubation period was uniform in all the inoculated fruits, in the irradiated fruits there was wide variability in the incubation periods. In the latter, the first signs of rot appeared within four days in a number offruits (8 per cent)
Stemphylium botryosum
0
4
8
12
d. AhffMrla
16 DAYS~TER?tN&“LAhON
tenuis
8121620X
-0 kmd 0 . . . ..-.. 0 CjOk& o----al00 o-.-.-o ~300
kmd 200 kmd kmd
Fxo. 1. The incubation period and percentage of rot in Spadona pears inoculated with irradiated spore suspensions of four fungi (fruit held at room temperature).
Sensitivity to gamma irradiationofjhgi pathogenicto pears
tion of the incubation period of P. expansum and B. cinerea with increased dosage (Figs. 2 and 3).
but incubation was prolonged up to 24 days in the remaining fruit. Although irradiation of spore suspensions of A. tenuis and S. botryosum resulted in some inhibition of the germination ability of the spores in vitro (Table l), it did not prevent rot development in the inoculated fruits (Fig. 1). However, a noticeable prolongation of the incubation period of S. bob-yosum in the fruit occurred with 200 krads-8-19 days vs. the 4-11 days for control fruit. The incubation period of A. tenuk was not significantly lengthened as a result of irradiation. The slight increase which occurred at the lower doses was not enhanced by increasing the dosage. (b) Irradiation of inoculated &it. Irradiation of inoculated fruits showed a gradual prolonga,
,
,
,
a. Penicillium
,
,
,
As with fruit inoculated with irradiated suspensions, the marked result of irradiation, which increased with the dose, was the staggering effect on the incubation period. However, once the rot had begun to develop, its growth rate was more or less constant (Fig. 2). Inhibition of pathogenicity was noticeable from 200 krad, but it was much more marked with P. expsum than with B. cinerea (Fig. 3). Although a noticeable prolongation of an average of ten days in the incubation period of P. expanwm was obtained with the 100~krad dose, noticeable rot control occurred only at 200 krad. With this dose, 2.5 per cent of the fruit began to rot within 17 days and rot control of 60 per ,
expansum
,
,
,
,
,
,
,
P
b. Batrytis cinerea P
-
0 krad
o----O
50
o----o100 *--q-3
581
krad kmd
200 kmd
-3COkrad
FIG. 2. The effect of gamma irradiation on the growth rate of rots of Spadona pears stored at 0%.
582
Rivka Barkai-Golan,Ruth Ben-Arie, Sylvia Gue~at-Reichand R. S. Kahan ,
Cl?,, ; ,’ : :
,
(,
0. Peniciltium
,
,
,
,
,
,
expansum
8 : ,I ;
:: s !f
-
<
0
b. Botrytis cinerea
0
6
16
W
0 krad
O-..-----0
50 kmd
O----0100
24 M’S
32 40 46 AFTER INOCi ILATlON
56
64
72
kmd
o-.-.-O
200 kmd
o----O
300 kmd
FIG. 3. The effect of gamma irradiation on the length of the incubation period and the percentage of rot development in inoculated Spadona pears stored at 0%. cent was achieved within two months’ storage. On the other hand, 50 per cent of the fruit inoculated with B. cinerea and irradiated with 300 krad, showed signs of rot within 33 days of inoculation.
DISCUSSION
AND CONCLUSIONS
P. expansum spores
in a suspension of 10s spores/ml entirely lost their germinating ability on a PDA medium following gamma irradiation with a lOO-krad dose, whereas they retained their ability to cause fruit rot. Also in fruit inoculated with a 104/ml spore suspension of P. expansum, the fungus developed after irradiation with 100 krad and some fruit was infected even following irradiation with 200 and 300 krad. However, it is important to note that, at
the time of irradiation, the fungus had already begun to invade the fruit tissues and irradiation therefore did not affect the freshly collected spores, but rather spores at various stages of germination. It was similarly found that, whereas a dose of 200 krad inactivated 99.8 per cent of B. cinerea spores in vitro in a suspension of lo3 spores/ml, all the fruit inoculated with the same suspension was infected. These results are in agreement with the higher lethal dose of gamma rays described by BERAHA~~) for mycelium of B. cinerea within the host, in comparison with artificial culture media; and also with the total inhibition of colonial growth of Botrytis in culture obtained by a 300-krad dose as compared to the ineffectiveness of the same dose in controlling
Sensitivity to gamma iwadziation offungi pathogenic to @us the pathogenicity of the fungus to strawberries under the same conditions(s). The in vivo experiments show that irradiation with gamma rays resulted in a noticeable prolongation of the incubation periods of both P. exparuum and B. cinerea-tie two most common causal agents of storage rots in Spadona pears. Even though partial control of both fungi was obtained only at relatively high doses, in nature the actual concentration of spores with which the fruit is in contact at harvest is much lower, and the effect of irradiation is likely to be more marked.@*“*s) On the other hand, doses exceeding 200 krad have been found to prevent normal ripening of this fruitos).
REFERENCES 1. BERAHA L., RAMSEY G. B., SMITH M. A. and WRIGHT W. R. Phytofiathology 49, 91 (1959). 2. BARKA&~L.AN R. and KAHAN R. S. Prelim. Refit. Nat. Univ. Inst. Agr. Rehovot, Israel, No. 511
(1965) (in Hebrew, with English summary). 3. SOMMERN. P., MAXIE E. C., FORTLAGER. J. and ECKERTJ. W. Rad. Bot. 4, 317 (1964).
583
4. BARKAI-GOLAN R. and KAHANR. S. Plant Dis. Rep. 50,874 (1966). 5. SARAVACOS G. D., HATZIPETROU L. P. and GEOROIADOUE. Food Zwad. 3, A6 ( 1962). 6. SOMMERN. P., MAXIE E. C. and FORTLAGER. J. Rad. Bot. 4,309 (1964). 7. SOPOLOV G. P., Radiobiologiya 4,460 (1964). 8. BARKAI-GOLAN R., TEMKIN-G~RODEISKIN. and KAHAN R. S. Food Iwad. 8, 34 (1967). 9. NELSON K. E., MAXIB E. C. and E~KEL W. Phytofiathology 49,475 (1959). 10. COUEY H. M. and BRAMLAGE W. J. Phytopathology 558, 1013 (1965). 11. BARKAI-GOLAN R. and KAHAN R. S. Prelim. Rep. Nat. Univ. Agr. Rehovot. Israel, No. 605 (1968) (in Hebrew, with English summary).
12. BARKAI-GOLAN R., KAHAN R. S. and TEMKINGORODEISKIN. 579 (1968).
13. BEN-AFUE R.
Int. J. a#.
Radiat.
Isotopes 19,
Alon Hanotea 25 (4), 189 (1968)
(in Hebrew).
14. BERAHA L., SMITH M. A. and WRIGHT W. R. Phytojathology 58,474 ( 1960). 15. GUELFAT-REICH S., BEN-ARIE R., KAHAN R. S. and EISENBERGE. J. Hart. Sci. 44 (2), (1969).