Microscopic and molecular detection of Leptosphaeria maculans and L. biglobosa ascospore content in air samples

Abstract

Airborne sexual spores of Leptosphaeria maculans and L. biglobosa are primary inoculum of fungi causing phoma stem canker of oilseed rape. In this study ascospore release of these two species was compared between Lithuania (of autumn 2010 and 2011) and Poland (autumns of 2009 till 2012), using identical equipment and methods. Dynamics of L. maculans and L. biglobosa ascospore dispersal was investigated using volumetric samplers followed by microscopic and molecular methods of detection. In Lithuania, the earliest detection of Leptosphaeria ascospores, using microscopy was on 1 st September (2010) and on 2 nd September (2011), whereas in Poland the earliest detection was on 5th September (2009 and and the latest on 11 th October (2011), which demonstrates that differences between the seasons may exceed a month (36 days). In the case of molecular detection performed for the Polish samples, the dates of the earliest and the latest release of the first ascospores ranged from 5 th September to 15 th October (40 days). The number of days with ascospores detected in the air samples ranged from 6 (2012) to 25 (2009). At earliest, the detection of the highest concentration of ascospores was on 6 th September (2010) and the latest -on 28 th October (2009). These results demonstrate big differences between the results of spore monitoring between the years and the importance of aerobiological studies to follow the development of the pathogen in natural infection. The fluctuations in quantities of spores and fungal deoxyribonucleic acid (DNA) strongly depended on the weather conditions, mainly rainfall, air temperature and relative humidity. Ascospore release was observed immediately after the rainfall events, but not later than two days after the rainfall. On days without rain, spores were present in the air samples when average relative air humidity exceeded 90% and day temperature was below 15°C. In 2009, molecular detection revealed the presence of both fungal species in the air samples, whereas in 2010-2012 only L. biglobosa DNA was found. Molecular detection with a quantitative real-time polymerase chain reaction (qPCR) was identified as a fast and accurate method of pathogen detection and identification from air samples. The highest coefficient of correlation between microscopic and molecular detection of Leptosphaeria species was 0.828 (2009) and the lowest was 0.483 (2011); in all years both techniques of detection were statistically correlated. We have demonstrated that the correlation coefficient highly depended on the number of ascospores in air samples. Based on the studies performed in Lithuania and Poland, we have shown that climatic conditions in the northern part of Central Europe are favourable for the spread of Leptosphaeria spp. by ascospores; however, the patterns of ascospore release greatly differ between the both countries.

Air pollution by allergenic spores of the genus Alternaria in the air of central and eastern Europe

“…Moreover, comparable correlation was observed also in respect to fungal spores of other phytopathogens, belonging to Leptosphaeria maculans - Leptosphaeria biglobosa species complex (Kaczmarek et al 2014). The studies undertaken in parallel in Lithuania and Poland also showed significantly less spores and shorter spore seasons in the north (Kedainiai district, Lithuania), as compared to experiment site located inland, in south-east Poland (Piliponyte-Dzikiene et al 2014). This example also supports the hypothesis of the primary importance of temperature on Alternaria spore production and release, over rainfall and relative humidity.…”

Aeromycology: studies of fungi in aeroplankton

“…Moreover, the Andersen impactor allows sieving the spores into groups of different sizes. The recognition of fungi present in the air are then based on traditional visual assessments (Kasprzyk & Worek 2006, Grinn-Gofroń & Strzelczak 2011, Stępalska et al 2012, Pusz et al 2013, as well as on molecular tools (Kaczmarek et al 2009, Piliponyte-Dzikiene et al 2014.…”

Monitoring blackleg (Leptosphaeria spp.) ascospore release timing and quantity enables optimal fungicide application to improved oilseed rape yield and seed quality

“…Wind-borne ascospores adhere to the leaf surface and germinate to produce germ tubes, which penetrate the leaf indirectly through stomata or wounds; endophytic mycelia of the pathogens then grow along the leaf petioles asymptomatically to cause lesions on main and branch stems (Hammond et al 1985). Monitoring of the presence and concentration of fungal spores in air samples makes it possible to identify periods of disease risk to plants (West et al 2002a;Piliponyte-Dzikiene et al 2014). The detection of airborne inoculum sources at early stages of plant infection allows to assist in decisions on the optimal time for fungicide application (Gladders et al 1998).…”

“…Similar studies were also done in Lithuania (Brazauskiene and Petraitiene 2006) as well as in the Czech Republic (Jedryczka et al 2010). Ascospores of L. maculans and L. biglobosa in air samples were being trapped using volumetric spore samplers and then quantified with light microscopic and molecular techniques (Jedryczka et al 2010;Kaczmarek et al 2012;Piliponyte-Dzikiene et al 2014).…”