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. 2023 Jul 4;12(13):2543.
doi: 10.3390/plants12132543.

Real-Time PCR to Phenotype Resistance to the Citrus Nematode Tylenchulus semipenetrans Cobb

Marta Ruiz  1 , Annie Du Vo  1 , J Ole Becker  2 , Mikeal L Roose  1
Affiliations

Real-Time PCR to Phenotype Resistance to the Citrus Nematode Tylenchulus semipenetrans Cobb

Marta Ruiz et al. Plants (Basel). .

Abstract

As pest management options, such as nematicides, become more restrictive, developing rootstocks resistant to the citrus nematode is fundamental for citrus production. This study provides an updated methodology to screen for citrus nematode resistance in rootstock-breeding programs. We developed a novel method to extract female citrus nematodes from roots that is suitable for molecular work and a real-time-PCR-based nematode quantification method for Tylenchulus semipenetrans. These procedures allow scaling up screening to high-throughput workflows, increasing the chances of finding rootstock candidates that combine all the desired traits. Our results contribute to the growing literature supporting quantification of nematodes with molecular methods.

Keywords: Poncirus; breeding; extraction; pest; plant parasitic; qPCR; quantification; rootstock; screening; trifoliate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Melting curve profile of Tylenchulus semipenetrans amplicons with the melting temperature at 83.5 °C. No amplification was observed in control reactions without T. semipenetrans DNA.
Figure 2
Figure 2
Standard curve of Ct values plotted against the log of the starting quantity of juvenile nematodes based on serial DNA preparation dilutions.
Figure 3
Figure 3
Standard curve of Ct values plotted against the log of the citrus nematode female starting quantity based on serial DNA preparation dilutions.
Figure 4
Figure 4
Relationship between the number of female nematodes added to the solution and the number of nematodes predicted from the Ct values using the model y = −4.41x + 32.01. Values are the means of 3 replicates and their standard error.
Figure 5
Figure 5
Correlation between the female nematode abundance counted on each host root and the abundance predicted from Ct values using the model y = −4.41x + 32.01. Green dots identify known resistant hosts, and red dots identify known susceptible hosts. Groups clustered within the red and green clouds were created with 2-step cluster analysis (SPSS). Values are the means of n = 9 and their standard error. Data are log (x + 1) transformed.
Figure 6
Figure 6
Nematode juveniles (J2) collected from the inoculum rearing on Argentina sweet orange (ASwo) and Carrizo citrange (CC) in a greenhouse after 27 days of root incubation. Data are the means of n = 3, bars represent the standard error, and letters indicate significant differences for the t-test at p < 0.05.
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References

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