Zentralbl. Mikrobiol. 146 (1991),419-424Gustav Fischer Verlag Jena

[Botany Department, Faculty of Science, Cairo University, Giza, Egypt]

Growth Activities of the Sugarbeet Pathogens Fusarium solani (Mart.)Sacc., Rhizoctonia solani Kuhn and Sclerotium rolfsii Sacco underPyramin Stress

M. S. EL-ABYAD and AMIRA M. ABu-TALEB

Key words: growth activity, sugar beet, Fusarium so/ani. Rhizoctonia so/ani. Sclerotrium rolfsii, pyramin,herbicide

Summary

Pyramin at the concentration of 25 ug . ml I exerted little or no effect on growth activities of the sugarbeetpathogenic fungi Fusarium so/ani, Rhizoctonia so/ani and Sclerotium rolfsii. Tolerance of these fungi to highconcentrations (;" I ,000 ug . ml" I) of the herbicide varied at the different stages of development. Macroconidialgermination and sporulation of F. so/ani occurred in a wide range of pyramin concentrations (25-2,000 ug .ml- I

) although they were consistently inhibited with the increases in herbicide concentration. In the absence ofherbicide, R. so/ani exhibited the fastest growth of the fungi studied. However, growth tolerance to pyramin washighest for F. so/ani and lowest for R. so/ani and S. rolfsii. Sclerotia of S. rolfsii were the most sensitive topyramin with regard to their germination and numbers produced.

Zusammenfassung

Das Herbizid Pyramin verursachte in Konzentrationen von 25 ug/rnl geringe oder gar keine Effekte auf dieWachstumsaktivitaten von Zuckerriiben-pathogene Pilze, wie Fusarium so/ani. Rhizoctonia so/ani and Sclero­tium rolfsii. Die Toleranz dieser Pilze gegeniiber hohen Konzentrationen (;" 1,000 ug/rnl) des Herbizidsvariierte in Abhangigkeit der unterschiedlichen Pilzentwicklungsstadien. Die Makrokonidienkeimung undSporulation von Fusarium so/ani wurde bei Pyramin-Konzentrationen zwischen 25 und 2,000 ug/rnl beobachtet,wobei mit zunehmender Herbizidkonzentraion auch eine zunehmende Wachstumshemmung einherging (imVergleich zur Kontrolle ohne Herbizidanwendung).

1mallgemeinen war die Wachstumstoleranz gegeniiber Pyramin bei F. so/ani am h6chsten und bei R. so/aniund S. rolfsii am niedrigsten. Die Sclerotien von S. rolfsii waren in bezug auf ihre Keimung und gebildetenAnzahl am empfindlichsten gegeniiber Pyramin.

Chloridazon (pyrazon) [5-amino-4-chloro-2-phenyl-3 (2H)-pyridazinone] is the activeingredient of the herbicide pyramin, one of the hetrocyclic herbicides that belong to thepyridazine group. Pyramin is used for the control of weeds in sugarbeet at rates of 1.5-2 kg/ha. This herbicide is reported to have inhibitory effects on many microorganisms (KASZUBIAK1966; VALASKOVA 1967; JENSEN 1969; ZHARASOV 1969) but little information is availablepertaining to its effects on plant pathogens.

Because pyramin is a commonly used herbicide in sugarbeet production in Egypt, thisstudy was counducted to examine the ranges of tolerance of the developmental stages of threesugarbeet pathogenic fungi to this herbicide. Since this investigation is of an ecological nature,a wide range of concentrations, including some that exceed field rates, have been utilized.

420 M. S. EL-ABYAD and A. M. ABu-TALEB

Materials and Methods

Sources and culture of fungi. Fungi used in this work: F. solani (Mart.) Sacc., R. solani Kiihn and S.rolfsii Sacc. were isolated from diseased sugarbeet roots. The latter two species are pathogenic to three

sugarbeet variaties: Kawepoly, Primahill and Monopur (EL-ABYAD et al. 1988a). F. solani is pathogenic toTrirave and Kaumera varieties (unpublished data). These fungi were maintained in culture on modified Czapek­Dox agar (ABEYGUNAWARDENA and WOOD 1957) which consisted of (gil): sucrose, 20; KN03 , 4; NaHzP04 , 2;MgS04 , 7H zO, I; KCI, 0.5; yeast extract, 0.02; micro-elements mixture, I ml; agar, 15 and distilled water, I I.

Herbicide. Stock solutions of pyramin were prepared with sterile distilled water and diluted for differentconcentrations (25, 100,200,400, 1,000 and 2,000 ug . ml" I medium) of active ingredient.

Soil. The soil used in growth experiments was clayey in texture and its chemical analysis was(mg· 100 g'l); PzOs, 3; KzO, 53; MgClz, 85; NaC!, 130; CaClz, 335 and pH 8. The other properties were:moisture content, 23 %: organic carbon, 0.76 %; total carbonates, 4.18 %; total-soluble salts, 0.28 %; loss-on­ignition, 10.67%.

Germination of macroconidia. These experiments were conducted employing microscope slides covered,each, with I ml of macroconidial suspension of F. solani in aqueous solution of the desired concentration ofherbicide in Petri-dishes. The dishes were incubated at 2rC for 9 h in complete darkness. The percentage ofgermination and average length of germ-tubes were assessed after EL-ABYAD et al. (1983).

Germination of sclerotia. It was found difficult to conduct experiments with sclerotia produced by R.solani as they were embedded in the mycelium and bound together by mycelial strands which made itsquantitative estimations hazardous. Sclerotia of S. rolfsii were surface sterilized by soaking for 5 min in 1:400(WIV) bromine in water to kill viable hyphal extensions, washed and dried (ABEYGUNAWARDENA and WOOD1957). Ten sclerotia/Petri-dish were placed on the surface of water agar (2% WIV) supplemented with pyraminto produce herbicide concentrations ranging from 25-2,000 fl . ml~ 1medium. The plates were incubated for 69h at 2rC and the percentage of germinated sclerotia was determined.

Dry weight yields. The medium described by RODRIGUEZ-KABANA (1969) was used in these experiments,these consisted of (gil): dextrose, 30; KHzP04, I; MgS04·7HzO, 0.5; KCI, 0.5; KN03 , 2; I ml of each of thestock solutions: FeS04·7HzO, MnS04·7HzO, ZnS04'7HzO, and thiamine; distilled water, I L. Sterilemedium was mixed with the herbicide to produce concentrations ranging from 25-2,000 ug :ml~ I medium anddispensed in 50 ml aliquots in 250 ml flasks. The flasks were each inoculated with a 6 mm agar disk bearingmycelium of the fungus from actively growing colonies and incubated for 9 days at 27°C. The mycelium washarvested, dried to constant weight at 80°C and the dry weight yield was recorded.

Growth in soil. The soil growth tube method (EL-ABYAD et al. 1983) was used. The tubes were filled withair-dried sieved soil and autoclaved for 20 min at 120°C. An aqueous solution of the desired concentration ofpyramin, or distilled water for the control, was added to bring the soil to 60')'0 of its moisture holding capacity.The tubes were inoculated with a I crrr' agar block of mycelium and incubated at 2rc. Growth was measuredafter 14 days or less depending on the species.

Production of macroconidia. Modified Czapek-Dox agar was mixed aseptically with pyramin in amountscalculated to produce the required concentration and poured into Petridishes. The dishes were inoculated with a6 mm disk of mycelium of F. solani, incubated for 9 days at 2rC and the number of spores produced wascounted by a haemocytometcr (EL-ABYAD et al. 1983).

Production of sclerotia. Plates of herbicide-supplemented media, prepared as in the production ofrnacroconidia, were inoculated with S. rolfsii and incubated for 9 days at 2rc. Numbers of sclerotia producedin every treatment per crrr' of colony were visually counted.

Statistics. The experiments were conducted in 3-4 replicates and the results obtained were treatedstatistically with an analysis of variance and the significance was expressed at the LSD I %2) and 5%1).

Results

Germination of macroconidia and sclerotia

Macroconidia of F. solani germinated in a wide range of pyramin concentrations(25-2,000 ug . ml- I

) , although the percentage germination steadily decreased with anincrease in herbicide concentration (Table I). Maximum inhibition was recorded at theconcentration 2,000 ug . ml- 1 with a decrease in the germination rate from 63.3% in theuntreated control to 12.8 'Yo in the herbicide variant. Similar results were obtained when

Growth Activities of the Sugarbeet Pathogens 421

Table I. Percentages germination (G%) of macroconidia of F. so/ani (after 9 h) andsclerotia ofS. rolfsii (after69h) in various concentrations of pyramin (ug:ml- I

) at 27"C. Average length of germtubes (Gt urn) ofmacroconidia and hyphal extensions (Lh urn) of sclerotia are alsopresented.

Pyramin coen. F. so/ani S. rolfsii([.lg·ml- I )

G('%) Gt (urn) G(%) Lh (urn)

0 63.3 12.1 59.6 3,825.225 58.4 8.52) 49.8') 1,778.02)

100 46.8') 6.8') 37.5') 504. ]2)

200 39.]2) 8.2') 29.0') 386.0')400 25.3') 7.7') 13.4') 19.4')

1,000 17.2') 6.4') 1.02) 5.1')2,000 12.8') 6.1') 0') 0')

LSD 1% 7.8 1.7 10.0 533.05% 5.8 1.3 7.4 391.0

I) = significant at LSD 5%; 2) = highly significant at LSD I %.

measuring the length of the germ-tube after 9 h incubation. Maximum reduction in germ-tubelength was also obtained at herbicide concentration of 2,000 ug . ml" t (from 12.1 to 6.1 urn).

Sclerotia of S. rolfsii were highly sensitive to pyramin. The germination of sclerotia wasconsistently inhibited with increasing herbicide concentrations from 59.6% in the control to49.8% in 25!!g' ml- 1

, to 37.5% in 100!!g' ml- l, to 29% in 200 ug : ml- l

, to 13.4% in400 ug . ml" I , and to 1% in 1,000 ug . ml" 1. Germination was prevented at 2,000 !!g . ml" I

pyramin. The average length of hyphal extensions was similar to germination (Table 1).

Mycelial growth

Dry weight estimations (Table 2) showed that mycelial tolerance to pyramin was highestfor F. solani and lowest for R. solani and S. rolfsii. For F. solani, the results recorded at theconcentrations 25, 100,200 and 400 ug . ml- l (323.9,336,343.8 and 342.4 mg, respecti­vely) were not significantly different from the control (357 mg). Highly significant reductionsin growth yields observed at concentration of 1,000 !!g . ml- 1 (from 357 to 174.7 mg) and2,000 ug . ml" t (from 357 to 124.1 mg). Growth of R. solani was not significantly affected bya pyramin concentration of 25 f.tg . ml" 1. However, dry weight yields were significantlyreduced from 508.4 mg in the control to 456.6 mg in 100 ug . ml" 1, to 415.9 mg in 200 f.tg .ml- l

, to 411.8 mg in 400!!g . ml- I, and to 217.7 mg in 1,000!!g' ml- l. No growth was

recorded at the concentration 2,000 ug . ml- I. S. rolfsii was more sensitive to the herbicide

than the other two species. The results indicated that growth was highly significantly inhibitedin all the treatments when compared to the control. Growth yields at 25, 100,200 and 400!!g .ml- t(314.8, 116.3,85.3 and 34.8 rng, respectively) were very low as compared to the control(481.9 mg). No growth was recorded at the concentrations 1,000 and 2,000 ug : ml"".

Results of growth in soil (Table 2) showed that in the absence of herbicide, R. solaniexhibited the fastest growth (143 mm in 12 days) followed by F. solani (55.5 mm in 14 days),whereas growth of S. rolfsii was limited (16 mm in 14 days). Growth after 14 days for F.solani at the concentrations of 25 and 100 !!g . g-I (45 mm each) was lower than the control(55.5 mm) at LSD 5%. Increasing the herbicide concentration above 100 f.tg . g-I causedsignificant retardations in growth from 55.5 mm at the control to 40 mm at200!!g' g-l, to 34 mm at 400!!g' g-I, to 28.5 mm at 1,000!!g' g-l and to 24 mm at

29 Zentralbl. Mikrobiol.. Bd. 146. 6

422 M. S. EL-ABYAD and A. M. ABU-TALEB

Table 2. Effects of various concentrations of pyramin on dry weight yields of mycelium (Dry wt mg) ofF. so/ani, R. so/ani and S. rolfsii after growing for 9 days in modified Czapek-Dox medium at 27°C. Alsopresented growth (mm) as measured by the soil growth tubes (Sg) after 14days or less.

Pyramin concn. F. so/ani R. so/ani S. rolfsii(ug-rnl I)

Dry wt Sg Dry wt Sg Dry wt Sg

0 357 55.5 508.4 143 481.9 1625 323.9 451) 499.1 114.5') 314.8') 20

100 336 451) 456.6') 70.5') 116.3') 14200 3438 40') 415.9') 702) 85.32) 12400 342.4 34') 411.8 2) 70') 34.8') 92)

1,000 174.7") 28.5') 217.7') 552) 0') 72)

2,000 1241') 24') 0 52.5') 02) 62)

LSD 1% 48.5 14.2 51.S 19.5 38.7 5.95% 36.2 10 38.3 13.8 28.6 4.1

I) = significant at LSD 5 %; 2) = highly significant at LSD I %.

2,00 ug . g-l. R. solani appeared to be more sensitive than F. solani to the herbicide. Thegrowth recorded in all the treatments were significantly lower than the control; the magnitudeof retardation was directly proportional to the concentration. S, rolfsii was the most sensitiveof the fungi tested to pyramin. A concentration of 25 ug . g-) appeared to be stimulatory,concentrations of 100 and 200 ug . g- I did not significantly affect growth. All otherconcentrations studied were significantly inhibitory (9 mm at 400 ug . g-), 7 mm at1,000 ug . g-l and 6 mm at 2,000 ug . g-I compared with 16 mm for the control).

Production of macroconidia and sclerotia

F. solani sporulated over a wide range of concentrations ofpyramin (25-2,000 ug . ml- I )

(Table 3). Sporulation at 25 !Jog . ml- 1 (344.4 X 104 macroconidia ml- I) was not significantly

Table 3. Numbers of macroconidia and sclerotia produced after 9 days incubation at 27 °C by F. so/ani andS. rolfsii respectively on modified Czapek-Dox agar supplemented with various concentrations of pyramin.

Pyramin concn. F. solani- S. rolfsii"(ug-rnl I)

0 314.7 3525 344.4 31

100 220.6 1) 7')200 ISO') 2')400 123.1') 0')

1,000 110') 0')2,000 95.6') 0')

LSD 1% 50.1 7.55% 37.1 5.4

a = numbers of macroconidia produced (X 104ml I). b = numbers of sclerotia produced (sq-crn""). ') =highly significant at I % LSD.

Growth Activities of the Sugarbeet Pathogens 423

different from the control (314.7X 104 ml- l) . Lower macroconidial counts were noted when

compared with the control at the high concentrations studied with 314.7 x 104 m1- 1 to220.6X 104 at 100 ug . ml- 1• to 150 X 104 at 200 ug . ml- J

, to 123.1 X 104 at 400 ug . mr', tolJOx 104 at 1,000 ug . ml- I and to 95.6x 104 at 2,000 I-tg . mr '.

Production of sclerotia by S. rolfsii was highly suppressed in presence of pyramin (Table3). The results obtained at the concentration of 25 ug . ml- 1 was not significantly differentfrom the control. The number of sclerotia produced at the concentrations 100 and 200 ug .m1- J(7 and 2 respectively) was highly significantly reduced compared with the control (35).No sclerotia produced at the concentration of 400 ug . ml- I

.

Discussion

One of the important stresses to which sugarbeet pathogenic fungi are subjected is theapplication of the herbicide pyramin for the control of weeds in sugarbeet. This herbicide mayinterfere with the ecological processes within the soil and hence affect pathogenesis by thesoil-borne pathogens. For the sugarbeet pathogens, the success in producing disease dependson their ability to survive adverse conditions, to sporulate abundantly, to germinate vigo­rously, and to establish a parasitic relationship with their hosts. An interference with any or acombination of these processes would reduce the disease potential.

The results obtained in this work indicated that a pyramin concentration of 25 ug . ml- I

usually exerted little or no effect on growth activities of the fungi studied. Generally, growthtolerance to pyramin was highest for F. solani and lowest for R. solani and S. roifsii.

For S. rolfsii, applications of different herbicides to cereals and other field crops, havebeen reported to conceivably interfere with or enhance its activity (CURL et al. 1967;RODRIGUEZ-KABANA et al. 1967, 1969, 1970; PEEPLES and CURL 1970; VYAS et al. 1986).The sensitivity of R. solani in the mycelial state to high concentrations of herbicides has beenalso observed (KARR et al. 1979; LEACH and MURDOCH 1985; YOUSSEF et al. 1985).

F. solani exhibited a wide-range of tolerance to pyramin. A similar behaviour was notedfor this species in presence of the herbicide trifluralin (MARCHISIO et al. 1979) althoughtrifluralin was reported to exert no effect to vegetative growth of F. solani f.sp. phaseoli whenapplied at field rate (WALKER and ALTMAN 1984).

Since tolerance of phytopathogenic fungi to stresses vary according to developmental stageof the fungus (EL-ABYAD et al. 1983, 1988b), it was necessary to examine the impacts ofpyramin on spore germination and sporulation of the fungi studied. The results revealed thatthe widest ranges of tolerance for germination of macroconidia and sporulation were exhibitedby F. solani. Sclerotia of S. rolfsii showed the narrowest ranges with regard to theirgermination and numbers produced. Suppression of production of sclerotia by S. rolfsii in thepresence of atrazine herbicide was reported (CURL et al. 1968). However, sclerotial initialformation was increased in soil treated with 1, I-dimethyl-3-(ex,ex,ex-trifluoro-m-tolyl) urea(fluometuron) at 1.5 and 10 I-tg . g-I soil (BOZARTH 1969). The sclerotia produced onfluometuron-treated medium were larger than those produced on the control medium(BOZARTH and TWEEDY 197\). Formation of sclerotia was also suppressed by 10 herbicides(VYAS et al. 1986).

Thus, it may be concluded that the fungi studied differed in their tolerance to highconcentrations of pyramin; F. solani exhibited the widest range of tolerance whereas S. rolfsiiand R. solani were sensitive. The mechanisms of tolerance and sensitivity will be examinedthrough studies of the impacts of pyramin on the metabolic activities of some of these fungi.

Acknowledgement

We are grateful to Professor M. KASSAS for reading the manuscript.

29'~

424 M. S. EL-ABYAD and A. M. ABU-TALEB

References

ABEYGUNAWARDENA, D. V. W., WOOD, R. K. S.: Factors affecting the germination of sclerotia and mycelialgrowth of Sclerotium rolfsii Sacco Trans. Brit. Mycol. Soc. 40 (1957),211-231.

BOZARTH, G. A.: Fluometuron degradation in soil and its effects on microorganisms. Ph. D. thesis, AuburnUniv., Auburn, Ala. (1969), 122 pp.

BOZARTH, G. A., TWEEDY, B. G.: Effect of pesticides on growth and sclerotial production of Scleroticumrolfsii. Phytopathol. 61 (1971), 1140-1142.

CURL, E. A., RODRIGUEZ-KABANA, R., FUNDERBURK JR., H. H.: Effect of dipyridyl and toluidine herbicideson growth response of Sclerotium rolfsii in soil. Phytopathol. 57 (1967), 6 (Abstr.).

- Influence of atrazine and varied carbon and nitrogen amendments on growth of Sclerotium rolfsii andTrichoderma virida in soil. Phytopathol. 58 (1968),323-328.

EL-ABYAD, M. S., ISMAIL, I. K., AL-MESHHADANI, S. A.: Effects of some biocides on Fusarium oxysporumformae speciales causing cotton and tomato wilts in Egypt. Trans. Brit. Mycol. Soc. 80 (1983),283-287.

EL-ABY AD, M. S., HINDORF. H., RIZK, M. A.: Impact of salinity stress on soil-borne fungi of sugarbeet. I.Pathogenicity implications. Plant and Soil 110 (l988a), 27-32.

- Impact of salinity stress on soil-borne fungi of sugarbeet. II. Growth activities in vitro. Plant and Soil 110(1988b),33-37.

JENSEN, H. L.: The effect of various herbicides on root nodule bacteria. Tidsskr. PIA. 73 (1969),309-317.KARR, G. W., GUDAUSKAS JR., R. T., DICKENS, R.: Effects of three herbicides on selected pathogens and

diseases of turfgrasses. Phytopathol. 69 (1979), 279-282.

KASZUBIAK, H.: The effect of herbicides on Rhizobium. I. Susceptibility of Rhizobium to herbicides. ActaMicrobiol. Pol. 15 (1966), 357-363.

LEACH, S. S., MURDOCH, C. W.: Evaluation of thiabendazole and pentachloronitrobenzene for the control ofthe Rhizoctonia disease complex on white potato (Solanum tuberosum L.). Amer Potato J. 67 (1985),459-469.

MARCHISIO, V. F., FONTANA. A., MOSCA, A. M. L.: The in vitro action oftrifluralin on Fusarium species. Atti.Acad. Sci. Torino CI. Sci. Fis. Mat. Nat. 113 (1979), 227-282.

PEEPLES, J. L., CURL, E. A.: Effect of the herbicide EPTC on growth and enzymatic activity of Sclerotiumrolfsii and Trichoderma viride. Phytopathol. 60 (1970),586 (Abstr.).

RODRIGUEZ-KABANA. R., CURL, E. A., and FUNDERBURK JR., H. H.: Effect of paraquat on growth ofSclerotium rolfsii in liquid culture and soil. Phytopathol. 57 (1967), 911-915.

RODRIGUEZ-KABANA, R.: Enzymatic interactions of Sclerotium rolfsii and Trichoderma viride in mixed soilculture. Phytopathol. 59 (1969),910-921.

RODRIGUEZ-KABANA, R., CURL. E. A.. FUNDERBURK JR., H. H.: Effect of trifluralin on growth of Sclerotiumrolfsii in liquid culture and soil. Phytopathol. 59 (1969), 228-232.

RODRIGUEZ-KABANA, R.. CURL, E. A., PEEPLES, J. L.: Growth response of Sclerotium rolfsii to the herbicideEPTC in liquid culture and soil. Phytopathol. 60 (1970). 431-436.

VALASKOVA, E.: The sensitivity of soil fungi to the effects of herbicides. Abstr. 6th Int. Congr. PI. Prot.,Vienna (1967), 606-607.

VYAS, S. c., AHMED, M., SINGH, R., SHASTRY, P. P.: Suppression of soil-borne pathogenes of soybean in vitroand in vivo by herbicides. Indian Phytopathol. 39 (1986), 395-399.

WALKER. G. B., ALTMAN . .I.: The influence of trifluralin on the bean root rot pathogen Fusarium solani. f.sp.phaseoli, Phytopathol. 74 (1984),814 (Abstr.).

YOUSSEF, B. A., AMR, A. M., HEITEFUSS, R.: Interactions between herbicides and soil-borne pathogens ofcotton under greenhouse conditions. Z. Pflanzenkrankh. Pflanzenschutz 92 (1985), 55-63.

ZHARASOV, S. U.: The effect of herbicides on the growth of micro-organisms. Vest. Sel-khoz, Nauki, Alma-Ata12 (1969), 94-98.

Authors' address: Prof. Dr. M. S. EL-ABYAD, Botany Department, Faculty of Science, Cairo University,Giza, Egypt.