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In vitro evaluation of Colombianplant extracts against Black Sigatoka(Mycosphaerella fijiensis Morelet)Jaime Niño a , Yaned M. Correa a & Oscar M. Mosquera aa Grupo de Biotecnología-Productos Naturales (GB-PN), Centro deInvestigación y Estudios en Biodiversidad y Recursos Genéticos(CIEBREG), Universidad Tecnológica de Pereira, A.A. 97, Pereira,ColombiaPublished online: 17 May 2011.
To cite this article: Jaime Niño , Yaned M. Correa & Oscar M. Mosquera (2011) In vitro evaluationof Colombian plant extracts against Black Sigatoka (Mycosphaerella fijiensis Morelet), Archives OfPhytopathology And Plant Protection, 44:8, 791-803, DOI: 10.1080/03235401003672939
To link to this article: http://dx.doi.org/10.1080/03235401003672939
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In vitro evaluation of Colombian plant extracts against Black Sigatoka
(Mycosphaerella fijiensis Morelet)
Jaime Nino*, Yaned M. Correa and Oscar M. Mosquera
Grupo de Biotecnologıa-Productos Naturales (GB-PN), Centro de Investigacion y Estudios enBiodiversidad y Recursos Geneticos (CIEBREG), Universidad Tecnologica de Pereira, A.A. 97,Pereira, Colombia
(Received 28 November 2009; final version received 21 January 2010)
In this work, 90 dichloromethane and methanol extracts obtained from 45 plantscollected at the Natural Reserve Bremen – La Popa (Colombia) and at theNatural Regional Park Ucumarı (NRPU, Colombia) belonging to five botanicalfamilies were evaluated at 1000 mg/l, for their in vitro fungicide activity throughthe ascospore germ tube elongation and the measurement of the mycelial radialgrowth of Mycosphaerella fijiensis assays. The methanol extracts from the speciesLycianthes acutifolia (Solanaceae) and Piper pesaresanum (Piperaceae); as well as,the dichloromethane extracts from P. pesaresanum and those from the Lauraceaefamily named Nectandra acutifolia and Ocoteca paulii, all inhibited M. fijiensisascospore germination in 100% in the germinative tube elongation assay. Withregards to the effects of the plant extracts on mycelial radial growth, the methanolextracts from P. pesaresanum and the dichloromethane one from N. acutifoliaboth showed 100% inhibition in this bioassay. Additionally, from thephytochemical screening on the dichloromethane and methanol extracts it wasfound that compounds such as alkaloids, phenols and terpenes were present inmost of the extracts evaluated and they might be the cause of the antifungalactivities reported.
Keywords: Antifungal; bioprospection; black leaf streak; crop protection;phytofungicides; plant pathogens
Introduction
Mycosphaerella is one of the biggest genus of the Ascomycete family, which enclosesnear 3000 species (Aptroot 2006) and near 7000 anamorphous species (Crous et al.2007).
Plantain and banana crops are affected by the fungus Mycosphaerella fijiensisMorelet which causes one of the most destructive diseases known as Black Sigatoka(BS) which generates great economic losses to farmers. The biggest damage causedby M. fijiensis infection to plantain and banana plantations are the reduction of thephotosynthetic capacity, due to foliar necrosis, producing bunches with low weight,quality, and early maturation, even under refrigeration; additionally, this diseaseaccelerates plantations aging and degeneration (Merchan 2002).
For BS control, contact and systemic fungicides are used. The contact orprotective ones (clorotalonil and mancozeb), remain over the plant surface, avoiding
*Corresponding author. Email: [email protected]
Archives of Phytopathology and Plant Protection
Vol. 44, No. 8, May 2011, 791–803
ISSN 0323-5408 print/ISSN 1477-2906 online
� 2011 Taylor & Francis
DOI: 10.1080/03235401003672939
http://www.informaworld.com
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pathogen invasion. In contrast, systemic fungicides (propiconazole, azoxystrobin)penetrate the leaves through the cuticle and can diffuse inside the plant throughvascular vessels. However, their applications increase production costs and causesignificant human health damages and environmental deterioration (Arzaulou 2008).
Although, the number of higher plants is considered to be superior to 250,000species, the number of them that have been studied for their phytoprotectiveactivities is reduced. However, higher plants contain natural products that can beused for agricultural pests and diseases control (Nduagu et al. 2008). Some plantextracts that have shown M. fijiensis fungicide activity are Matricaria sp.(Asteraceae), Annona muricata (Annonaceae) and Melaleuca alternifolia (Myrtaceae)(Jimenez et al. 2006). Okigbo and Emoghene (2004) evaluated the aqueous plantextracts from Azadirachta indica (Meliaceae), Ocimun gratissimun Linn. (Labiatae)and Vernonia amygdalina Del. (Asteraceae) at 10, 25, 50 and 100%; they found thatO. gratissimun extract at 100% inhibited completely M. fijiensis ascosporegermination. Marın et al. (2006) concluded that extracts from A. indica, Swingleaglutinosa (Rutaceae), Salvia officinalis (Lamiaceae) and the mixture of S. glutinosaand A. indica inhibited fungus growth.
Even more, Osorio-Salamanca (2006) found that the ethanolic extract of Sennareticula (Fabaceae) showed high in vitro protective activity against BS and arguedthat compounds such as polyphenols, coumarines, quinones, saponins, triterpenesand/or flavonoids might be related to the antifungal activity showed against BS.Likewise, the extract from M. charantia (Cucurbitaceae) showed in vitro activityagainst M. fijiensis (Polanco et al. 2004).
It has been found that many M. fijiensis species are resistant to differentfungicides used to control them in commercial plantations (Marın et al. 2003), as arethe cases of demethylation inhibitors (DMI) (Sierotzki et al. 2000) and the Qorespiration inhibitors (QoIs) (Grasso et al. 2006), among others. Therefore, thediscovery of new agents that can be used effectively against M. fijiensis, the causalagent of this disease in plantain and banana crops with good efficacy to control BS isof paramount importance. That is the reason why the GB-PN, continuing with thebioprospection studies on the flora from natural reserves in the Colombian coffeegrowing region, evaluated 45 dichloromethane and 45 methanol extracts in thesearch for antifungal agents that can be used effectively against M. fijiensis.
Materials and methods
Plant material
Aerial parts from 45 plants belonging to the botanical families Lauraceae,Piperaceae, Ranunculaceae, Rubiaceae and Solanaceae were collected at NaturalReserve Bremen – La Popa (Filandia-Circasia, Quindıo, Colombia) and at theNatural Regional Park Ucumarı (NRPU, Pereira, Risaralda, Colombia).
Plants were collected in August 2005 and specimens were classified by F.J.Roldan, a sample of each collected plant was deposited at the University ofAntioquia Herbarium (HUA, Medellın, Colombia).
Extraction of plant material
From the collected plants, the dichloromethane and methanol extracts wereobtained, according to the procedure described by Nino et al. (2006).
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Plant extracts phytochemical screening
The main secondary metabolites present at dichloromethane and methanol extractswere characterised by thin layer chromatography (TLC) by using Silica Gel 60 F254
sheets (Merck, Darmstadt, Germany). The system chloroform–ethyl acetate–methanol (2:2:1) and hexane–ethyl acetate (7:3) were used for methanol anddichloromethane extracts elution, respectively; the extracts were searched for thepresence of alkaloids, sterols, terpens, saponins, phenols, tannins and flavonoids,respectively (Wagner and Bladt 1996). All determinations were done in duplicates.
Evaluation of plant extracts against M. fijiensis
Ascospore germ tube elongation method
BS infected plantain leaves with in five and six stages of the infection (Belalcazar1991) were collected at Quimbaya (Quindıo, Colombia). From these leaves, 2 cm2
pieces were stapled to Kraft paper disks and incubated by 48 h inside a humidchamber at room temperature. After this, the disks were submerged in distilled waterfor 5 min and fixed to the internal surface of each Petri dish lid. The dishes werecovered and the ascospores were allowed to discharge during 1 h, onto the solidifiedagar (2%) surface, which was amended with each plant extracts at 1000 mg/l. Then,Petri dishes were incubated between 25 and 278C, during 24 and 48 h for methanoland dichloromethane extracts, respectively. After incubation, 150 ascospores(divided into 3 visual fields of 50 ascospores each) were evaluated in two differentPetri dishes per plant extract by using a light microscope with 610 amplification. Bythis reading, the number of ascospores with normal germination (G), amorphousgermination (AG), short germination (SG) and those that not germinated (NG) wasobtained (Du Pont 1983). All experiments were repeated at least twice at differenttimes.
Radial mycelial growth
A colony from a 14-days old monosporic culture and 5 ml sterile water and someglass beds were transferred to a test tube. The test tube was shaken for 30 s at2500 rpm in a vortex. Then, 100 ml of this suspension were transferred and spread onthe agar surface contained on Petri dishes amended with 250 mg/l of streptomycinand the appropriate volume to make a final concentration of 1000 mg/l for eachplant extract analysed. The radial growth of 15 different colonies (five colonies inthree different Petri dishes) was read. Readings on the same colonies were performedat days 7, 9, 12, 15 and 20 of the incubation period. The dishes were incubated at25–278C under darkness (Pelaez et al. 2006).
Data analysis
The extracts with antifungal activity in the sexual phase were evaluated in relation tothe emerging germ tube length of ascospores as G, AG, SG and NG; while, theasexual phase was determined by measuring the mycelial radial growth during 20days. Both assays were arranged in a completely randomised design replicated twice.Data on both bioassays were analysed by using the Infostat software version 2008I.
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In addition, the mycelial radial growth data were evaluated through a non-parametric analysis of Kruskal–Wallis, with a confiability range of 95% by using thesame software.
Results and discussion
A list of the collected plants with their respective registration numbers and the resultsof the phytochemical screening are shown in Table 1.
Germinative tube elongation of M. fijiensis ascospores
Figure 1 shows the most active methanol extracts in the germinative tube elongationof M. fijiensis ascospores assay. The methanol extracts from Lycianthes acutifolia(UTP-3, Solanaceae) and Piper pesaresanum (UTP-148, Piperaceae), inhibitedascospores germination in 100 and 96%, respectively, expressed as NG (Figure 1).
In addition, the methanol extracts from the species Piperomia acuminata (UTP-154, Piperaceae), Solanum sp. (UTP-161, Solanaceae) and Rodostemonodaphne sp(UTP-162, Lauraceae) showed SG of M. fijiensis ascospores with values of 83, 95and 95%, respectively. Even more, the methanolic extracts from the Solanaceaefamily, named Solanum ovalifolium (UTP-51) and Solanum deflexiflorum (UTP-100)displayed simultaneously amorphous (AG) and NG M. fijiensis ascospores withadditive values of 100%.
The antifungal activity showed by the members of the Solanaceae family can berelated to saponins and steroidal glycoalkaloids typically synthesised by some genusof this important plant family. To these, secondary metabolites have been reported awide range of biological and pharmacological activities such antifungal, antibacter-ial, antiparasitic, cytotoxical, antitumor, among others (Sparg et al. 2004; Devkotaet al. 2008).
The dichloromethane plant extracts that displayed the highest inhibitory actionagainst the germinative tube elongation of M. fijiensis ascospores are shown inFigure 2.
The dichloromethane plant extracts from the Lauraceae family named Nectandraacutifolia (UTP-177) and Ocoteca paulii (UTP-179) as well as the one fromP. pesaresanum (UTP-148, Piperaceae) inhibited M. fijiensis ascospore as NG in100% (Figure 2). These results are in consonancy with the facts that many speciesfrom the Lauraceae family and also from the genus Ocoteca are important sources oflignans with a wide range of biological activities, such as antineoplastic,antimicrobial, antifeedant, among others (Da Silva-Filho et al. 2007). Moreover,from many Ocoteca species have been isolated unusual lignans, neolignans andalkaloids with important biological activities (Guerrini et al. 2006; Garcez et al.2007). In consequence, secondary metabolites like lignans or alkaloids could beresponsible for the antifungal activities showed by N. acutifolia (UTP-177) andO. paulii (UTP-179), since from the phytochemical screening developed in this work,these types of natural products were detected on the methanolic extracts of theseplants.
The fungicide action of both the methanol and dichloromethane plant extractsthat caused high amount of amorphous germ tubes on M. fijiensis might have similarmechanism of action to those one displayed by Benlate1 (1%), the positive controlused on these experiments. For this reason, the methanol extracts from
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Table
1.
Phytochem
icalscreeningof90extractsfrom
plants
collectedin
theNaturalReserveBremen-LaPopa(Q
uindıo,Colombia)andtheNatural
RegionalPark
Ucumarı(N
RPU,Risaralda,Colombia).
Family
Scientificname/
voucher
FJR
/UTPnumber
Secondary
metabolitesb
Extract
aAlkaloids
Steroids
andtriterpens
Saponins
Phenols
Flavonoids
Tannins
Lauraceae
Ocoteainsulares4031/176
CH
2Cl 2
7þ
7þ
7þ
MeO
H7
77
77
7Rodostem
onodaphnesp
4011/162
CH
2Cl 2
7þþ
þþ
þþ
þþþ
MeO
Hþþ
77
7þþ
7Nectandra
acutifolia4032/177
CH
2Cl 2
7þ
7þ
7þ
MeO
H7
77
þþ
7þþ
Nectandra
lineatifolia4033/178
CH
2Cl 2
7þ
7þ
7þ
MeO
H7
77
þþþ
þOcoteapaulii4034/179
CH
2Cl 2
þþþ
þ7
þþ
þþþ
MeO
Hþþþ
77
77
7
Piperaceae
Piper
pesaresanum
3996/148
CH
2Cl 2
þþþ
þ7
þ7
þMeO
Hþþþ
77
þþ
þþþ
Peperomia
acuminata/154
CH
2Cl 2
þþ
77
þþ
þMeO
Hþþ
þþþ
þþþ
þþþ
þPiper
eriopodon/158
CH
2Cl 2
þþ
7þ
7þ
MeO
Hþþ
77
77
7Piper
umbellatum
4012/163
CH
2Cl 2
þ7
77
77
MeO
H7
77
77
7Piper
crassinervium
4021/167
CH
2Cl 2
77
77
þþ
7MeO
H7
þþ
þþ
þþþ
þþ
þþþ
Piper
glanduligerum
4026/172
CH
2Cl 2
77
þþþ
þþþþ
þMeO
Hþþ
77
7þ
7Piper
sp/175
CH
2Cl 2
77
77
þþ
7MeO
H7
þþ
þþ
þþþ
þþ
þþþ
Piper
calceolarium
4048/194
CH
2Cl 2
7þ
þþ
þþ
MeO
H7
þþ
þþ
þPiper
daniel-gonzalezii4051/197
CH
2Cl 2
7þ
þþ
7þ
MeO
Hþ
þþ
þþ
þ
(continued)
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Table
1.
(Continued
).
Family
Scientificname/
voucher
FJR
/UTPnumber
Secondary
metabolitesb
Extract
aAlkaloids
Steroids
andtriterpens
Saponins
Phenols
Flavonoids
Tannins
Ranunculaceae
Thalictrum
podocarpum
3991/144
CH
2Cl 2
þþþ
þþ
7þþ
þþþ
MeO
Hþþþ
77
þþþþ
þClematishaenkeana4005/156
CH
2Cl 2
7þþ
þþ
þþ
7þþ
MeO
H7
þþþ
þþþ
77
7
Rubiaceae
Cinchonapubescens3161/8
CH
2Cl 2
77
77
77
MeO
Hþ
77
77
7Hoffmannia
asperula
3169/16
CH
2Cl 2
77
77
77
MeO
Hþ
7þ
7þ
7Palicoureapetiolaris3182/28
CH
2CL2
77
þ7
77
MeO
Hþ
7þ
77
7Palicoureaandaluciana3183/29
CH
2Cl 2
77
77
77
MeO
Hþ
7þ
þ7
7Palicoureathyrsiflora
3184/30
CH
2Cl 2
77
77
77
MeO
Hþ
7þ
þ7
7Dioicidendrondiocum
3748/79
CH
2Cl 2
7þ
77
77
MeO
H7
þþ
77
7Guettardacrisipiflora
4023/169
CH
2Cl 2
7þ
þþþ
þ7
þMeO
H7
77
77
7
Solanaceae
Lycianthes
radiata
3154/1
CH
2CL2
77
þ7
77
MeO
H7
77
77
7Witheringia
coccoloboides
c3155/2
CH
2Cl 2
77
77
77
MeO
Hþ
77
77
7Lycianthes
acutifolia3156/3
CH
2Cl 2
77
77
77
MeO
Hþ
7þ
77
7Solanum
sp3173/20
CH
2CL2
77
þ7
77
MeO
Hþ
7þ
77
7Solanum
ovalifolium
Dunalc3714/
51
CH
2Cl 2
7þ
77
77
MeO
Hþ
77
þ7
7
(continued)
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Table
1.
(Continued
).
Family
Scientificname/
voucher
FJR
/UTPnumber
Secondary
metabolitesb
Extract
aAlkaloids
Steroids
andtriterpens
Saponins
Phenols
Flavonoids
Tannins
Solanum
deflexiflorum
Bitter3718/
55
CH
2Cl 2
7þ
77
77
MeO
Hþ
þ7
7þ
7Lycianthes
synantheraBitter3719/
56
CH
2Cl 2
7þ
77
77
MeO
H7
77
7þ
7Depreaglabra
A.T.Hunziker
3722/
59
CH
2Cl 2
7þ
77
77
MeO
H7
7þ
7þ
7Browallia
speciosa
Hookc3732/60
CH
2Cl 2
7þ
77
77
MeO
H7
77
77
7Solanum
stellatiglandulosum
3744/
77
CH
2Cl 2
7þ
77
77
MeO
Hþ
77
þþ
7Solanum
ochranthum
3922/101
CH
2Cl 2
þþ
þþ
þþ
MeO
Hþ
þþ
þþ
þDunaliasolanacea3992/145
CH
2CL2
7þ
7þ
7þ
MeO
Hþþ
77
7þ
7Lycianthes
radiate
3993/146
CH
2Cl 2
7þ
þþ
þþ
7þþ
MeO
Hþþþ
77
þ7
þSolanum
sp4010/161
CH
2Cl 2
þþ
þþ
þ7
þMeO
Hþþþ
þþ
þþ
7þ
7Solandra
coriacea4013/164
CH
2Cl 2
7þ
7þ
7þ
MeO
H7
þ7
7þ
7
(continued)
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Table
1.
(Continued
).
Family
Scientificname/
voucher
FJR
/UTPnumber
Secondary
metabolitesb
Extract
aAlkaloids
Steroids
andtriterpens
Saponins
Phenols
Flavonoids
Tannins
Witheringia
coccoloboides
c4019/
165
CH
2Cl 2
þþ
þþ
þþ
þþ
7þþ
MeO
Hþþ
77
þþ
þCestrum
humboldtii4022/168
CH
2Cl 2
7þþ
þþþ
7þþ
MeO
Hþþ
77
77
7Depreaaffsachapapa4024/170
CH
2Cl 2
7þþþ
þþþ
7þþ
MeO
Hþþ
777
þ7
þBrowallia
speciosa
c4025/171
CH
2Cl 2
7þþ
7þþ
7þþ
MeO
H7
þþþ
þþþ
77
7Solanum
ovalifolium
c4027/173
CH
2Cl 2
7þ
7þ
7þ
MeO
H7
77
77
7Solanum
deflexiflorum
Bitter3921/
100
CH
2Cl 2
þþ
77
77
MeO
Hþ
þþ
þþ
7Solanum
brevifolium
4028/174
CH
2Cl 2
7þþ
7þþ
7þþ
MeO
Hþþ
þþ
77
77
Positive
controls
Papaverine
Hecogenin
and
stigmasterol
Dioscin
Aof
protosaponin
Resorcinol
Kaem
pferol
Tannic
Acid
þþ
þþ
þþ
aCH
2Cl 2,dichloromethane;
MeO
H,methanol.
bþ,presentatlow
quantities;þþ,presentatmoderatesquantities;þþþ,presentathighquantities;7,absent.
ccollectedatdifferenttimeandplaces.
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S. deflexiflorum (UTP-100), T. podocarpum (UTP-144) and G. crisipiflora (UTP-169);as well as the dichloromethane one from H. asperula (UTP-16), Rodostemonodaphnesp. (UTP-162), W. coccoloboides (UTP-165), B. speciosa (UTP-171), S. ovalifolium
Figure 1. Active methanol extracts against M. fijiensis in the germ tube elongation assay.
Figure 2. Active dichloromethane extracts against M. fijiensis in the germ tube elongationassay.
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(UTP-173) and S. brevifolium (UTP-174) could be considered as potential fungicideagents as all showed amorphous germ tubes development similar to Benlate.
Radial growth results
The methanol extracts that showed the highest fungicide activity against M. fijiensison the mycelial radial growth are shown in Figure 3. The methanol extract fromP. pesaresanum (UTP-148, Piperaceae) showed 100% of inhibition in this bioassay;followed by the methanolic extracts of W. coccoloboides (UTP-2, Solanaceae),Cinchona pubescens (UTP-8) and Palicourea andaluciana (UTP-29) that displayed 70,70 and 65%, of inhibition in this bioassay, respectively.
The dichloromethane extracts that showed the highest fungicide activity againstM. fijiensis mycelial radial growth are shown in Figure 4. Those extracts fromN. acutifolia (UTP-177, Lauraceae), as well as those from the Piperaceae family
Figure 3. Methanol extracts with activity in the mycelia radial growth assay of M. fijiensis.
Figure 4. Dichloromethane extracts with inhibition on the mycelial radial growth ofM. fijiensis.
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P. pesaresanum (UTP-148) and P. acuminata (UTP-154) all displayed the highestpercentage of inhibition in this bioassay, with values of 98, 78 and 70%, respectivelyagainst this phytopathogenic fungus.
The highest fungicide activity displayed by the dichloromethane and methanolextracts against M. fijiensis was shown by P. pesaresanum (UTP-148, Piperaceae)since both extracts inhibited the fungus normal development in its sexual and asexualreproductive phases and these actions can be attributed to alkaloids, sterols andphenolic compounds evaluated through the phytochemical screening (Table 1).These types of secondary metabolites have been reported consistent andconstitutively in the Piperaceae family (Parmar et al. 1997) and in particular onthe genus Piper (Facundo et al. 2004; Campos et al. 2005; Lago et al. 2005).
According to Nam et al. (2004), the in vitro fungitoxic activities from the extractsdepend on several factors related to the phytocompound constituents, among themare included the natural products intrinsic activity, their lipophilicity, theincorporation percentage as well as the cellular metabolism rate. In general, theplant extracts analysed in this work showed different grades of activities dependingupon the botanical family where each extract originates and this predicts the specificextract constitution. From the phytochemical screening, the dichloromethaneextracts were abundant in steroids, triterpens and saponins; while, the methanolones were rich in alkaloids, saponins, flavonoids and tannins. In general, thevariations in the antifungal effects of the plant extracts evaluated are related to thequantitative differences in their secondary metabolite constituents.
It could be considered that phytocompounds present in the active extracts act onthe sexual and asexual phases of M fijiensis reproductive cycle by inhibiting thegrowth of the germinative tube length, the mycelium radial growth or by multiplemechanisms, perhaps, similar to those followed by known synthetic fungicides; forexample, by inhibiting: the sterol biosynthesis (Ma and Michailides 2005), the sterolD7/D8-isomerase (Pennati et al. 2006), the fungi mitotic division (Nakaune andNakano 2007), among others. In addition, plant extract secondary metabolites mightblock specific gene expression related to the synthesis of small to high molecularweight agents associated to the phytopathogenicity of M. fijiensis.
Therefore, it is important to conduct research that allows the isolation andidentification of secondary metabolites from P. pesaresanum, N. acutifolia andO. paulii with an in vitro antifungal activity against M. fijiensis and to study themechanism of action of the isolated compounds.
Conclusion
Among the several plant extracts evaluated in this work, those from P. pesaresarum,N. acutifolia and O. paulii showed very promising results to be used in M. fijiensiscontrol. In addition, this work proves that it is feasible to discover novel bioactivenatural products, which can be used in plantain and banana crop protection againstBS, and can add value to the regional flora and contribute to their preservation.
Acknowledgements
The authors are very grateful to the Centro de Investigacion y Estudios en Biodiversidad yRecursosGeneticos (CIEBREG), TheUniversidadTecnologica de Pereira andCOLCIENCIASfor the financial support to this project. In addition, the authors are also in debt with theCARDER and CRQ corporations by for granting permission to plant collection.
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