Development of a PCR-based diagnostic assay for the specific detection of the entomopathogenic fungus Metarhizium anisopliae var. acridum

Entz, Susan C.; Johnson, Dan L.; Kawchuk, L. M.

doi:10.1017/s0953756205003746

Cited by 39 publications

(32 citation statements)

References 40 publications

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“…Moreover, molecular identification of potential EPF is gaining acceptance as an important first step for successful development of myco-insecticides (Islam et al, 2014). Particularly, molecular tools such as PCR based analysis of DNA are used as standard procedures for identification and phylogenetic comparisons between EPF (Jensen et al, 2001;Destefano et al, 2004) and as molecular markers for species identification (Driver et al, 2000;Entz et al, 2005;Islam et al, 2014). The objective of this study was, therefore, to identify and evaluate the potentials of native entomopathogenic fungi for the development of myco-insecticides against P. interrupta.…”

Section: Sorghummentioning

confidence: 99%

Molecular characterization and evaluation of indigenous entomopathogenic fungal isolates against Sorghum Chafer, Pachnoda interrupta (Olivier) in Ethiopia

Habtegebriel¹,

Getu²,

Dawd³

et al. 2016

J. Entomol. Nematol.

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Sorghum chafer, Pachnoda interrupta (Olivier) is a damaging pest of sorghum, other cereals and horticultural crops in Africa which results in complete crop loss. Currently, the management strategies rely heavily on chemical pesticides, which do not provide effective control. There is evidence showing that microbial biocontrol agents have the potential to control P. interrupta. In this study, the entomopathogenic fungi isolated from soils and insects in Ethiopia were identified using morphological and cultural characteristics. These were then evaluated for viability and virulence against Galleria mellonella (Fabricius) (Lepidoptera, Pyralidae) larvae and P. interrupta adults. Firstly, 116 Metarhizium spp. and Beauveria spp. were considered. The isolates were evaluated based on high viability as measured by percentage germination on SDA media. Only 56 isolates had greater than 70% viability. Secondly, these were further evaluated using conidial suspensions applied to G. mellonella larvae at a concentration of 1×10 8 conidia/ml. Four Beauveria spp. and three Metarhizium spp. isolates which caused over 60% mortality were selected for final virulence assay against adults of P. interrupta. The selected isolates were further confirmed by PCR amplification of ITS4 and ITS5 gene regions and chi1 and chi4 primers. Finally, the seven isolates were evaluated for virulence against P. interrupta at a dose of 1 mg dry conidia/10 beetles under laboratory condition. Mortality of P. interrupta ranged from 14% for isolate 9604 to 82% for isolate PPRC51. Dose-response tests showed that the LD 50 of PPRC2 (0.62 mg/10 beetles) and PPRC51 (0.55 mg/10 beetles) isolates were not significantly different from each other. The results demonstrated the high potential of the two isolates (PPRC51 and PPRC2) as microbial biocontrol agents. However, field evaluation of the isolates should be performed for their development into a mycopesticide against P. interrupta.

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Section: Sorghummentioning

confidence: 99%

Molecular characterization and evaluation of indigenous entomopathogenic fungal isolates against Sorghum Chafer, Pachnoda interrupta (Olivier) in Ethiopia

Habtegebriel¹,

Getu²,

Dawd³

et al. 2016

J. Entomol. Nematol.

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“…However, these technical approaches are usually laborious and not sensitive enough, and do not provide objective and quantitative results. During the past three decades, a PCR-based strategy has been developed and used for evaluation of fungal biomass within plant tissue (Bretagne et al, 1995;Entz et al, 2005;Färber et al, 1997;Jurado et al, 2006;Nicholson et al, 1997). Although this technique has many advantages due to its broad spectrum, simplicity, sensitivity and time-and labourefficiencies, it is basically qualitative and not quantitative because the amount of amplicon does not reflect the copy number of template DNA if the amount of each sample's amplified DNA has reached saturation level prior to the checking point.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Alternaria brassicicola infection in the Arabidopsis thaliana and Brassica rapa subsp. pekinensis

Su’udi

Park

et al. 2013

Microbiology

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Black spot caused by Alternaria brassicicola is an important fungal disease affecting cruciferous crops, including Korean cabbage (Brassica rapa subsp. pekinensis). The interaction between Arabidopsis thaliana and Alt. brassicicola is a representative model system, and objective estimation of disease progression is indispensable for accurate functional analyses. Five strains caused black spot symptom progression on Korean cabbage and Ara. thaliana ecotype Col-0. In particular, challenge with the strains Ab44877 and Ab44414 induced severe black spot progression on Korean cabbage. Ab44877 was also highly infective on Col-0; however, the virulence of Ab44414 and the remaining strains on Col-0 was lower. To unveil the relationship between mycelial growth in the infected tissues and symptom progression, we have established a reliable quantification method using real-time PCR that employs a primer pair and dual-labelled probe specific to a unigene encoding A. brassicicola SCYTALONE DEHYDRATASE1 (AbSCD1), which is involved in fungal melanin biosynthesis. Plotting the crossing point values from the infected tissue DNA on a standard curve revealed active fungal ramification of Ab44877 in both host species. In contrast, the proliferation rate of Ab44414 in Korean cabbage was 3.8 times lower than that of Ab44877. Massive infective mycelial growth of Ab44877 was evident in Col-0; however, inoculation with Ab44414 triggered epiphytic growth rather than actual in planta ramification. Mycelial growth did not always coincide with symptom development. Our quantitative evaluation system is applicable and reliable for the objective estimation of black spot disease severity.

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“…anisopliae (Destéfano et al, 2004) and M. anisopliae var. acridum Driver, Milner & Trueman (Entz et al, 2005). The ISSR technique was used in studying the genetic variability of two varieties of M. anisopliae from different localities (Lima, 2005) and Beauveria bassiana (Balsamo) Vuillemin in China (Wang et al, 2005), Asia (Aquino de Muro et al, 2005) and Japan (Takatsuka, 2007).…”

Section: Introductionmentioning

confidence: 99%

Polymorphism in Metarhizium anisopliae var. anisopliae (Hypocreales: Clavicipitaceae) based on internal transcribed spacer-RFLP, ISSR and intron markers

Tiago¹,

Carneiro-Leão²,

Lima³

et al. 2011

Genet. Mol. Res.

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ABSTRACT.Isolates of entomopathogenic fungus Metarhizium anisopliae var. anisopliae were characterized using internal transcribed spacer-RFLP, ISSR and intron splice site primers. Thirtyseven isolates were studied, most of which were obtained from the sugar cane pest, Mahanarva fimbriolata (Hemiptera: Cercopidae) from Tangará da Serra, Southwest Mato Grosso State, Brazil. Internal transcribed spacer-RFLP did not differentiate the isolates of M. anisopliae var. anisopliae, while ISSR and intron primers identified three distinct groups. Variability among these groups was 96% for (GTG) 5 and 100% for the other primers. We found considerable genetic variability, even among isolates from the same geographical origin and host.

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Development of a PCR-based diagnostic assay for the specific detection of the entomopathogenic fungus Metarhizium anisopliae var. acridum

Cited by 39 publications

References 40 publications

Molecular characterization and evaluation of indigenous entomopathogenic fungal isolates against Sorghum Chafer, Pachnoda interrupta (Olivier) in Ethiopia

Molecular characterization and evaluation of indigenous entomopathogenic fungal isolates against Sorghum Chafer, Pachnoda interrupta (Olivier) in Ethiopia

Quantification of Alternaria brassicicola infection in the Arabidopsis thaliana and Brassica rapa subsp. pekinensis

Polymorphism in Metarhizium anisopliae var. anisopliae (Hypocreales: Clavicipitaceae) based on internal transcribed spacer-RFLP, ISSR and intron markers

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