Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

Abstract

Flow cytometers are probably the most multipurpose laboratory devices available. They can analyse a vast and very diverse range of cell parameters. This technique has left its mark on cancer, human immunodeficiency virus and immunology research, and is indispensable in routine clinical diagnostics. Flow cytometry (FCM) is also a well-known tool for the detection and physiological status assessment of microorganisms in drinking water, marine environments, food and fermentation processes. However, flow cytometers are seldom used in plant pathology, despite FCM's major advantages as both a detection method and a research tool. Potential uses of FCM include the characterization of genome sizes of fungal and oomycete populations, multiplexed pathogen detection and the monitoring of the viability, culturability and gene expression of plant pathogens, and many others. This review provides an overview of the history, advantages and disadvantages of FCM, and focuses on the current applications and future possibilities of FCM in plant pathology.

Coffee rust genome measured using flow cytometry: does size matter?

“…Despite the common use of FCM for the measurement of genome size in plants and animals, the application of this technique in fungi has been minimal. The genome size measurement of fungi is relatively difficult, as they often have complicated life cycles with ploidy levels ranging from 1 × to 50 × and the basic chromosome numbers are often unknown (D'Hondt et al ., ). Moreover, in rust fungi with thick‐walled spores, such as H. vastatrix , this morphological characteristic represents a crucial technical problem for obtaining sufficient nuclei for FCM analyses because isolated nuclei have to be used for the stoichiometric staining reaction and the DNA quantitative measurements.…”

“…(), the nuclear content of the standard should be similar to that of the sample being analysed. The current study adopted this strategy by using S. lycopersicum , because there are no stable and well‐characterized fungal internal standards (D'Hondt et al ., ). In fact, as shown by Bennett et al .…”

“…Genome size analysis has gained increasing attention in both plant and animal studies, as a result of more accurate and efficient quantification techniques (Gregory et al ., ). Knowledge of the nuclear DNA content has considerable potential for use in plant pathology, offering valuable insights into the cytology, pathogenicity and phenology of the pathogens involved (D'Hondt et al ., ).…”

“…Flow cytometry (FCM) is a technique much used in the animal and plant kingdoms that ‘has become the method of choice for ploidy and genome size determination because it is fast, cheap and easy’ (D'Hondt et al ., ). Nevertheless, FCM has been little exploited to determine genome size of the Kingdom Fungi, despite the fact of its proven accuracy (D'Hondt et al ., ). However, difficulties in the use of FCM procedures have been encountered with some fungal groups and their analogues, especially biotrophic, nonculturable plant pathogens, such as the rust fungi (Pucciniales).…”

Flow cytometry reveals that the rust fungus, Uromyces bidentis (Pucciniales), possesses the largest fungal genome reported—2489 Mbp

“…Genome size in Eukaryotes varies greatly, as a result of polyploidization and/or structural chromosomal rearrangements. Genome size and its dynamics have direct implications on the evolutionary fitness, reproduction and non-sexual mechanisms for diversity creation (D'Hondt et al, 2011). Among Fungi, previous studies have revealed that genomes vary from <1 to 893 Mbp (Kullman et al, 2005;Tavares et al, 2014).…”