Accuracy and precision of population estimates of Verticillium dahliae on growth media in quantitative soil assays

Abstract

Verticillium dahliae Kleb. is a serious pathogen of many plant species. Growth media used to measure population density of V. dahliae in soil were evaluated for high recovery of the pathogen, as well as accuracy and precision of population estimates from naturally and artificially infested sandy loam soil using the soil dilution method. Recovery of V. dahliae from naturally infested field soil was highest on soil pectate Tergitol agar (SPT), soil extract agar + sodium polypectate (SEAP), modified pectate agar (MPA), potato dextrose agar + streptomycin sulphate (PDAS), and Talboys' prune lactose agar (TPA); however, PDAS and TPA were overgrown with contaminating fungi, making enumeration difficult. Use of SPT medium resulted in the most precise population estimate with a standard error (SE) of 12.6% of the mean followed by use of pectate agar (PA) (SE = 14.8%) and SEAP (SE = 19.1%). Ethanol, biotin, and Dox salts enhanced recovery of V. dahliae from naturally infested soil, but combining Dox salts with ethanol and biotin significantly reduced population density. Soil extract had no significant effects on population density. Accuracy of V. dahliae population estimates from sterile artificially inoculated soil was highest on modified soil extract agar (MSEA) (64%) followed by SPT (58%). However, accuracy of V. dahliae population estimates from nonsterile artificially inoculated soil was highest on SPT (36%). Soil extract is not an essential ingredient and biotin may increase recovery of V. dahliae from soil. Résumé :Le Verticillium dahliae Kleb. est un virulent pathogène chez plusieurs espèces de plantes. Les milieux de culture utilisés pour mesurer la densité des populations du V. dahliae dans le sol, ont été analysées en utilisant la mé-thode de dilution de sol, afin de déterminer le maximum de détection du pathogène, ainsi que l'exactitude et la préci-sion des estimés de population de sables loameux, naturellement ou artificiellement infectés. La quantité de propagules du V. dahliae détectées à partir de sols de prairie naturellement infectés est plus élevée sur les géloses à base de sol et de pectate tergitol (SPT), extrait de sol gélosé avec polypectate de sodium (SEAP), pectate gélosé modifié (MPA), gé-lose avec dextrose et pomme de terre additionnée de sulfate de streptomycine (PDAS), et gélose de Talboy à base de lactose de prune (TPA); on note que les PDAS et TPA sont envahis par des contaminants fongiques qui rendent l'énumération difficile. L'utilisation du SPT fournit les estimations de populations les plus précises, avec une déviation standard (SE) de 12,6 % par rapport à la moyenne, suivi de l'utilisation du PA (SE = 14,8 %) et du SEAP (SE = 19,1 %). L'éthanol, la biotine et les sels Dox augmentent la détection du V. dahliae à partir de sols naturellement infectés, mais la combinaison des sels Dox avec l'éthanol et la biotine diminue significativement la densité de population. L'exactitude des estimations des populations du V. dahliae, à partir de sol stérilisés et inoculés artificiellement, est plus é...

Multiplex Real-Time Quantitative PCR to Detect and Quantify Verticillium dahliae Colonization in Potato Lines that Differ in Response to Verticillium Wilt

“…Nevertheless, Q-PCR amplifications of the two samples of infected potato tissues showed higher consistency of quantification compared with CFU counts. Similar to other related studies, the variability between the two samples tested by CFU counts was high (35,41), with the standard deviation on the CFU counts reaching 346 with a mean of 212 (data not shown). Conversely, Ct values from both DNA samples using Q-PCR fluctuated by ±0.5, which is within the normal variation of the iCycler iQ system, and a similar range of variation observed among the three amplification replicates from each DNA sample.…”

“…NPX plates are incubated for at least 2 weeks and the medium allows for the growth of other species in the genus Verticillium (e.g., V. albo-atrum and V. tricorpus), which complicates pathogen quantification. Another complicating factor lies in the stochastic nature of the quantification using CFU counts, where a high variability within replicates of one sample commonly is observed (41). Furthermore, competition among colonies may affect V. dahliae quantification on culture media.…”

Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo‐atrum

“…This is important for the development of disease prediction models as well as for the validation of control strategies. Many of those detection methods rely on culturing the fungus on selective media (Mpofu and Hall, 2003; Termorshuizen et al ., 1998). However, these methods are time‐consuming, laborious and require extensive taxonomic expertise (Termorshuizen et al ., 1998).…”

Scientific opinion on the pest categorisation of Verticillium dahliae Kleb

“…dahliae can also be detected in soil, by mean of sieving and wet or dry plating (Smith, 1965;Goud et al, 2003). Recovery of V. dahliae from naturally infested field soil was highest on soil pectate tergitol agar (SPT), soil extract agar + sodium polypectate (SEAP), modified pectate agar (MPA), potato dextrose agar + streptomycin sulphate (PDAS) and Talboys' prune lactose agar (TPA) (Mpofu and Hall, 2003). Termorshuizen et al (1998) carried out an interlaboratory comparison of methods used for the quantification of V. dahliae in soil and concluded that the wet plating assays give lower number of propagules and are less accurate than the dry plating assays.…”