Effect of potassium bicarbonate on fungal growth and sclerotia of Sclerotium cepivorum and its interaction with Trichoderma

Blanca Lilia Ortega-Aguilar Alejandro Alarcón Ronald Ferrera-Cerrato

Área de Microbiología, Postgrado de Edafología, Colegio de Postgraduados, Campus Montecillo 56230, Estado de México. México 1 1

Efecto del bicarbonato de potasio en el crecimiento fúngico y esclerocios de 0 2

,

Sclerotium cepivorum y su interacción con Trichoderma 1 6 - 3 5

Resumen. Se estudió el efecto del bicarbonato de potasio (KHCO3) como agente antifúngico : 3

en el crecimiento de los hongos y cepa R39. Se evaluó el 3

Sclerotium cepivorum Trichoderma A crecimiento del micelio, el número y germinación de esclerocios formados por S. Í G

, así como el antagonismo de sobre el patógeno, en el medio de O cepivorum Trichoderma L

® O

cultivo papa dextrosa agar (PDA, Baker ) enriquecido con siete concentraciones de KHCO C

3 I M

(2, 4, 6, 8, 10, 25 and 50 mM). Con 50 mM de KHCO3, el crecimiento de S. cepivorum fue E

inhibido en 34.5% y el de en 83.1%. Con la aplicación de 10, 25 y 50 mM de D Trichoderma A

KHCO3, la formación de esclerocios por S. cepivorum fue significativamente inhibida N A C

(51.5%, 77.0% y 100%, respectivamente), al igual que la germinación de los esclerocios I X

(18%, 78% y 86%, respectivamente). Al confrontar ambos hongos en el medio de cultivo E M enriquecido con KHCO , no se afectó la capacidad antagónica de hacia el

3 Trichoderma A T S

patógeno. Después de 144 h, Trichoderma mostró una colonización del 70% sobre el micelio I V E de S. cepivorum, ante 50 mM. Los beneficios potenciales del KHCO3 para inhibir tanto el R

crecimiento y desarrollo de S. cepivorum fueron evidentes al reducir el crecimiento fúngico,

/

la formación y germinación de esclerocios.

o

Palabras clave: compuesto antifúngico, antagonismo, fitopatógeno, biocontrol. c i x é M

n

Abstract. The effect of potassium bicarbonate (KHCO3) as an antifungal agent was tested on e

a the fungal growth of and strain R39. The growth of the s Sclerotium cepivorum Trichoderma e r fungal colony, the number and germination of sclerotia formed by , as well as p S. cepivorum m I

.

the antagonism of on the pathogen were evaluated in potato dextrose agar a Trichoderma í ® g o (PDA, Baker ) culture media amended with seven concentrations of KHCO3 (2, 4, 6, 8, 10, l o c 25 and 50 mM). At 50 mM, the growth of was inhibited 34.5%, whilst for i S. cepivorum M

e

inhibition was 83.1%. At the concentrations of 10, 25 and 50 mM of KHCO d Trichoderma 3 a n

sclerotia formation by S. cepivorum was significantly inhibited (51.5%, 77.0% and 100%, a c i x

respectively), likewise the sclerotia germination (18%, 78% and 86%, respectively). When e M

both fungi were confronted in a KHCO -enriched PDA, this chemical compound did not a 3 t s i

affect the antagonistic capability of towards the pathogen. After 144 h, v Trichoderma e R

1

Trichoderma showed an invasion of 70% over the colony of S. cepivorum, at 50 mM. The 1 0 2 potential benefits of KHCO3 to inhibit both growth and development of S. cepivorum were

evident, since it reduced either fungal growth or both formation and germination of sclerotia. © Keywords: antifungal compound, antagonism, plant pathogen, biocontrol.

Received 18 July 2010; accepted 18 April 2011. Recibido 18 de julio 2010; aceptado 18 de abril 2011.

Autor para correspondencia: Alejandro Alarcón L

[email protected] A N I G I R O lasting agents for controlling pathogenic fungi such as and the Trichoderma strain was selected due to its A completely randomized experimental design was L A N Introduction I

Phytophthora, Sclerotinia, Sclerotium, Rhizoctonia, antagonistic and mycoparasitic ability against a wide range of used for the experiment that included eight treatments with G I R

F u s a r i u m , a m o n g o t h e r s ( Ts a h o u r i d o u a n d species of pathogenic fungi of cultivated plants, including S. five replicates each (n=5). Data were analyzed using analysis O Mexico at the end of 2010 had 19,225 ha cultivated with onion Thanassoulopoulos, 2002; Vinale et al., 2008; Ibarra-Medina cepivorum (unpublished data). of variance and the mean comparison test (Tukey, á=0.05) (Allium cepa L.), and average yield of 15,933 ton ha-1 (SIAP, et al., 2010), by producing toxic metabolites, inhibiting or Disks of medium and mycelium (0.9 cm of diameter) using SAS statistical program (SAS Institute Inc, 2002). 2011). However, this crop is affected by white rot, caused by parasitizing mycelium of soil-borne fungi (Zago et al., 2001). of either Trichoderma sp. R39 or S. cepivorum were placed at the fungus Sclerotium cepivorum Berk, which spreads rapidly On the other hand, bicarbonates are commonly used the center of Petri dishes containing Potato Dextrose Agar Phase III. Effect of potassium bicarbonate on the m

® u and causes significant crop losses (Pinto et al., 2000; Ponce- in food industry to avoid fermentation, control pH, and (PDA, Baker ) enriched with seven concentrations of KHCO3 antagonism in vitro of Trichoderma R39 on Sclerotium r o v i

Herrera et al., 2008). This fungus forms sclerotia, which may develop adequate textures or flavors (Karabulut et al., 2001; (2, 4, 6, 8, 10, 25, and 50 mM) whose pH was 7.0, 7.0, 7.1, 7.3, cepivorum p e c

remain in the soil for up to 20 years. The occurrence of the Bombelli and Wright, 2006). Their efficiency has also been 7.4, 7.9 and 8.0, respectively. Five Petri dishes were used for Petri dishes containing PDA and KHCO3 at the concentrations m u i t

disease is related to the presence and abundance of sclerotia in proven in the control of many phytopathogenic fungi (Palmer each concentration of KHCO3. PDA-dishes cultures without and pH indicated in the previous section were inoculated with o r e l c soil, since these structures remain dormant in the absence of et al., 1997; Gamagae et al., 2003). The bicarbonate ion has KHCO3 were used as control (pH 6.5). Fungal cultures were PDA disks with mycelium. Trichoderma R39 and Sclerotium S

f o the crop, and its germination is stimulated by root exudates of been identified as the probable cause of growth inhibition in incubated at room temperature (20°C) for four days, and the cepivorum were placed in opposite sides of the Petri dishes. a i t o

Allium species, which contain volatile compounds such as some bacteria and fungi. In the case of fungi, hyphal walls fungal growth of each fungus was measured each 24 h. Sclerotium cepivorum was inoculated first, due to its slow r e l c s

allyl sulfide and n-propyl (Entwistle et al., 1982; Tariq and collapse in the presence of bicarbonate, then causing either Afterwards, the Petri dishes with S. cepivorum were kept at growth, and Trichoderma R39 was placed 24 h later. Fungal d n

Magee, 1990). growth inhibition or fungal death (Palmer et al., 1997; room temperature (20°C) for 7 days (168 h), to count new- cultures were incubated at 20 °C and the growth of both fungi a

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In order to reduce the damage caused by this plant Ordoñez-Valencia et al., 2009). formed sclerotia at each concentration of KHCO3. was measured every 24 h for four days. The invasion w o r g

pathogen, several methods have been used, but the most There are reports about the effectiveness of A completely randomized experimental design was percentage of Trichoderma over the fungal colony of the l a g

common is the chemical control. However, the excessive use potassium bicarbonate (KHCO3) on the reduction of foliar used for the experiment which included eight treatments for pathogen was estimated according to the following equation n u f

of fungicides has caused pollution problems like leaving damage produced by fungi, as well as the benefit of each fungus with five replicates each (n=5). Data were (Rollan et al., 1999): C=[(DCAP/DSP)x100], where: n o

e t

residues in the harvested crop, and most important it has also Trichoderma species for controlling soil-borne fungi analyzed using analysis of variance and the mean comparison C=Invasion percentage; DCAP=distance covered by the a n o b

induced pathogen resistance (Fushiwaki et al. 1990; Ma and (Tsahouridou and Thanassoulopoulos, 2002; Vinale et al., test (Tukey, á=0.05) using SAS statistical program (SAS antagonist on the pathogen colony over the axis which r a c i 1

Michailides, 2005; Klose et al., 2010; Van Dyk and Pletschke, 2008). However, the effect of KHCO3 on the growth of S. Institute Inc, 2002). separates both fungi, DSP=distance between sowing points b

1 0 m 2 2011). Therefore, less environmentally harmful control cepivorum and on its interaction with Trichoderma is (6.5 cm). u i

, s s 3 a

3 alternatives have been sought. For example, biological unknown. This study evaluated the effect of KHCO on the Phase II. Effect of potassium bicarbonate germination of A completely randomized experimental design was 3 t

o A p Í

f

G control which is based on the use of antagonistic growth and development of S. cepivorum, and on its sclerotia of Sclerotium cepivorum used for the experiment that included eight treatments with o

O t L interaction with Trichoderma strain R39. Sclerotia of S. cepivorum were collected from Petri dishes c O microorganisms and on the use of innocuous chemical five replicates each (n=5). The data obtained were analyzed e f C f I E

M compounds, such as bicarbonates (Quiroz-Sarmiento et al., without KHCO from the previous experiment. Sclerotia were using analysis of variance and the mean comparison test 3 .

l E a

t D

2008; Ordóñez-Valencia et al., 2009; Ibarra-Medina et al., surface disinfected with 80% alcohol for 30 seconds, (Tukey, á=0.05) using SAS statistical program (SAS Institute e

A . L N 2010). The aim of these alternatives is to contribute on soil Materials and methods followed by several rinses with sterile distilled water. The Inc, 2002). . A B

C , r I a X

conservation, as well as to ensure quality food sources for sclerotia were transferred to a gentamicine solution (20 µg l i E u

M -1 g humans and to maintain a healthy environment (Bombelli and Phase I. Effect of potassium bicarbonate on the mycelial mL ) and incubated at 4°C for 24 h. Excess of antibiotic A A - T a S g I Wright, 2006). growth of Trichoderma and Sclerotium cepivorum solution was removed by blotting dry sclerotia on sterile e V t r E

R Laboratory and greenhouse studies have Cultures of Trichoderma sp. strain R39 and Sclerotium paper. Ten sclerotia were placed in each Petri dish containing O

successfully proven the effects of microbial antagonists on cepivorum were obtained from the Departamento de PDA and KHCO3 at the concentrations and pH described in plant pathogens (Arzate-Vega et al., 2006). In this way, Microbiologia, Colegio de Postgraduados. Sclerotium the previous section. Plates were incubated at 20°C, and thus, species of the genus Trichoderma are efficient and long- cepivorum was isolated from a garlic crop field at Guanajuato, germinated sclerotia were counted daily during seven days. 5 4 5 5 comparison to the control (Figure 1B). In contrast, after 96 h, germination was not observed (Figure 3). After 168 h, 100% L A N Results I

the application of 2, 10 and 25 mM, caused fungal growth germination of sclerotia was observed for concentrations G I R

inhibition of 1.5%, 5.8% and 6.4%, respectively; however, at between 0 and 8 mM, while at 10, 25 and 50 mM, germination O Phase I. Effect of potassium bicarbonate on the mycelial 50 mM the fungal growth reduction was 34.5% in comparison was 80%, 22% and 14%, respectively (Figures 3 and 4). growth of Trichoderma and Sclerotium cepivorum with the control (Figure 1B).

Concentrations of KHCO3 produced gradual increases on the KHCO3 caused significant (P<0.001) reductions on Phase III. Effect of potassium bicarbonate on the pH in culture media (from 7.0 to 8.0) when compared to the the number of sclerotia. After 168 h, fully formed sclerotia antagonism in vitro of Trichoderma R39 on Sclerotium m u

control (pH=6.5). After 24 h, concentrations of 2 and 6 mM of were observed in the concentrations from 0 to 10 mM; in cepivorum r o v i

KHCO3, significantly (P<0.001) inhibited the growth of contrast, at 25 mM the sclerotia formation just started, while Concentrations of KHCO3 up to 6 mM resulted in significant p e c

Trichoderma by 20.6% and 50.3%, respectively; the sclerotia did not form at 50 mM (Figure 2). At the differences (P<0.001) in the antagonistic capability of m u i t

concentrations of 10 mM and 25 mM caused an inhibition of concentrations of 2 and 10 mM of KHCO3, the number of Trichoderma towards the pathogen (Figure 5). After 96 h, o r e l

58.1% and 84.2%, while in 50 mM the growth of the fungal sclerotia decreased 9.1% and 51.5% respectively, while at 25 invasion of Trichoderma on the colony of S. cepivorum at c S

f o culture was entirely inhibited (Figure 1A). After 96 h, and 50 mM, the inhibition on the number of sclerotia was concentrations of 0 to 8 mM was approximately of 58%. At 10 a

Figure 3. Effect of increasing concentrations of potassium i t

bicarbonate (KHCO3) on the sclerotia germination of Sclerotium o concentrations of 2 and 6 mM inhibited fungal growth by 77.0% and 100% respectively, in comparison to the control and 25 mM the average percentage invasion of Trichoderma r e

cepivorum at 24, 48, 72, 96, 120, 144, and 148 h of exposure. l c

Means ± Standard error. n=5. s 4.0% and 7.3% while concentrations of 10, 25 and 50 mM (Figure 2). was 45%, while at 50 mM was 30% (Figure 5). After 120 h, d n

caused an inhibition of 15.7%, 17.5 and 67.9%, respectively the antagonism of Trichoderma on S. cepivorum persisted at a

h t

in comparison to the control (Figure 1A). Phase II. Effect of potassium bicarbonate germination of concentrations from 0 mM (control) to 25 mM, in which the w o r g

After 24 h, concentrations greater than 2 mM sclerotia of Sclerotium cepivorum average invasion percentage was 78% (Figure 5). At 50 mM, l a g

significantly (P<0.001) reduced the fungal growth, in KHCO3 significantly inhibited (P<0.001) the germination of n u f

sclerotia. After 24 h, sclerotia germination percentage was n o

e

t

26% and 20% in the control (0 mM) and in the concentration a n o b

of 2 mM, respectively. After 48 h, sclerotia germination at r a c i 1

control, 2 and 4 mM was 46%, 36% and 12% germination, b

1 0 m 2 respectively, whilst from concentrations from 6 to 50 mM, u i

, s s 3 a 3 t

o A p Í

f G o

O t L c O e f C f I E

M .

l E a

t D

e

A . L N . A B

C , r I a X l i E u M g

A A - T a S g I e V t r E

R O KHCO3 (mM) KHCO3 (mM) Figure 1. Effect of increasing concentrations of potassium Figure 2. Effect of increasing concentrations of potassium

bicarbonate (KHCO3) on the individual growth of Trichoderma bicarbonate (KHCO3) on the formation of sclerotia by Sclerotium R39 (A) and Sclerotium cepivorum (B). Means±Standard error. cepivorum, after 168 h. Means±Standard error. n=5. Figure 4. Effect of increasing concentrations of potassium bicarbonate (KHCO3) on the sclerotia germination of Sclerotium cepivorum, n=5. after 168 h. 6 7 5 5 y L n the invasion percentage was 53% (Figure 5) although the of bicarbonate in comparison to S. cepivorum. This inhibitory The growth of Trichoderma was more sensitive to 50 o A l o ) N c I % ( e pathogen showed less growth than Trichoderma (Figure 6). effect can be explained partly due to the pH changes that mM of KHCO3 than S. cepivorum in single cultures. In G I h t m R r u r O e After 144 h, Trichoderma grew over the colony of the bicarbonate concentrations induced in the culture media, thus contrast, when both fungi were confronted, the growth of S. o v i t o o a r e

l pathogen by 96% at concentrations from 0 to 10 mM, while at affecting the acidic requirements for fungal growth (Steyaert cepivorum was more limited. This effect can be in part m r c e s d a o i 25 and 50 mM, the invasion of the antagonist on S. cepivorum et al., 2010). For instance, Harman and Taylor (1994) explained due to the fungal antagonism in combination with h n i c t i r o r T was 80% and 70%, respectively (Figure 5). indicated that a pH 4.5 was more suitable for the growth of the increased pH in the culture media by KHCO . In this e 3 f l o c S n f

o Trichoderma sp. in vitro, whilst at pH of 2.0 and/or 8.0 the regards, Trichoderma species preferably grow up in acid i o s m a u v fungal growth is slow. The antifungal effect of KHCO environments than in alkaline ones (Benitez et al., 2004; r n 3 I o v i

Discussion observed on S. cepivorum is in accordance to findings that Steyaert et al., 2010), and their antagonistic capabilities may p e c

show the growth inhibition of S. rolfsii and Botrytis cinerea be influenced by the pH in the culture media (Bell et al., 1982; m u i t

Figure 5. Effect of increasing concentrations of potassium Results show that KHCO3 exerted significant effects on the under in vitro conditions, when bicarbonates were applied at Vinale et al., 2008). Likewise, the enrichment of culture o r e bicarbonate (KHCO3) on the invasion of Trichoderma (expressed l c growth of Trichoderma R39 and S. cepivorum. For 20 or 40 mM (Palmer et al., 1997; Corral et al., 1998; media with KHCO3 may result in modifications on growth in percentage) on the colony of Sclerotium cepivorum under in vitro S

f

conditions. Identical letters on bars at each sampling time are not o Trichoderma R39, the concentration of 50 mM inhibited in Bombelli and Wright, 2006). patterns of the fungal colony. For example, Trichoderma at a

significantly different (Tukey, =0.05). Means ± Standard error. i t

n=5. o 67.9% the fungal growth. In contrast, the growth of S. This study also shows that the number of sclerotia concentrations from 0 to 25 mM had greater sporulation, r e l c s

cepivorum at 50 mM was inhibited in 34.5%. These results formed by S. cepivorum was significantly inhibited at whereas S. cepivorum, exposed to 50 mM showed less d n

denote that Trichoderma was more sensitive to the presence KHCO -concentrations higher than 10 mM. Punja and abundance of mycelia, which has finer filaments (data not a 3 h t

Grogan (1982) and Palmer et al., (1997) found that the shown). w o r g

carbonates and bicarbonates applied in a concentration of 50 Our results agree with those from Tsahouridou and l a g

mM inhibited the formation and germination of sclerotia Thanassoulopoulos (2002) and De Souza et al. (2008) in the n u f

formed by S. rolfsii and B. cinerea. The decrease in the sense that Trichoderma species have antagonistic effects n o

e t

number of sclerotia observed in this study can be related to the against pathogens such as S. rolfsii and Moniliophthora a n o b

pH increase due to KHCO3. In this sense, several findings perniciosa. There are no earlier reports about the inhibitory r a c i 1

indicate that the pH affects both growth and sclerotia effects of KHCO3 on the antagonistic capability of b

1 0 m 2 formation by fungal phytopathogens such as B. cinerea, S. Trichoderma sp. on S. cepivorum, therefore the importance of u i

, s s 3 a

3 rolfsii, Sclerotinia sclerotiorum and Rhizoctonia solani (De this study. t

o A p Í

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G Pasquale and Montville, 1990; Ordóñez-Valencia et al., An inhibitory effect of KHCO3 on the formation and o

O t L 2009). germination of sclerotia at concentrations up to 10 mM was c O e f C f I E

M In addition, the present research shows the effects of observed. An 80% inhibition in sclerotia germination of S. .

l E a

t D

KHCO3 on the fungal confrontation between Trichoderma sp. rolfsii in the presence of 40 mM of KHCO3 was reported e

A . L N and S. cepivorum. The antagonic effect of Trichoderma on S. (Ricker and Punja, 1991). This effect shows the potential of . A B

C , r I a X cepivorum was maintained in all KHCO3-concentrations, but bicarbonates to control not only the growth of S. cepivorum l i E u M g the pathogen showed more growth inhibition at 50 mM. but also to inhibit the germination of sclerotia. In this regards, A A - T a S g I Although the antagonism of Trichoderma species on fungal Wilson (1999) showed that carbonates and bicarbonates e V t r E

R pathogens is well documented (Vinale et al., 2008), the in (sodium and potassium) are effective chemical compounds O vitro confrontation of this antagonist with S. cepivorum against B. cinerea which is the causal agent of postharvest

under culture media amended with KHCO3 has not been diseases in grapes. Likewise, applying 50 mM of ammonium Figure 6. Effect of increasing concentrations of potassium bicarbonate (KHCO3) on the antagonism of Trichoderma (Tri) on Sclerotium cepivorum (Sc), after 120 h. previously reported. 9 8 5 5 60 REVISTA MEXICANA DE MICOLOGÍA 33, 2011 Bell, Arzate-V reviewers CONACYT mM). This ef both bicarbonate bicarbonate cepivorum with sclerotia cepivorum Colletotrichum al KHCO d f KHCO produce while r bicarbonate olfsii o e fect ., r m m 2000; o D.K., a 25 fungi, work n on t Palmer under i 3 3 ega, s . o t and References Acknowledgements Conclusions Mexicana negra Mycosphaer Santos-Eméstica, had sclerotia n r by S. Gamagae for The H.D. a

was and t J., cepivorum

had (NH was e nevertheless a S. 50 58594. in in invaluable d n negative et del A.C.

d W cepivorum significantly T negative gloesporiodes significant mM vitr / 4 ells, f al. o richoderma the de HCO financially plátano o r at ella r

Fitopatología

et o Michel-Aceves,

(1997)

C.R. Sincere g of concentrations conditions al culture f e , fijiensis 3 u ) 2006. r even ., KHCO ef n comments reduced m ( Musa 2003). g Markhman, T fects

ef i was a richoderma ef n indicated Antagonismo fects l inhibited a

at fects R39 supported Morelet, thanks t , 24:98-104. media i sp.) p 3 o the inhibited Penicillium . on l n the a (Punja Sclerotia V

during n in in of .M. and highest t 1982. o the number

greater vitr vitr agente to f that as

p increased. bicarbonates Domíngez-Márquez,

thorough de a o kept s the and o growth the by t p the In by e T h . B. o richoderma germination concentration o The causal invernadero. r vitr spp. two concentration of g confrontation the e than 77% Grogan, its ciner s e o

sclerotia n

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antagonistic s revisions. antagonism h de grant anonymous (Korsten of

50 a ea and Potassium v s la

spp. u e both on mM did

sigotoka c 1982), Revista 100% h SEP- b of en sobre

O.A. e (50 not the e a of of of of S. S. S. et of n s

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