cereal seeds must meet several quality criteria , in particular good germinative potency ( 85% after 8 days ) and sanitary state ( absence of claviceps purpurea ) .
more generally , low contamination from fungi such as fusarium , septoria , or alternaria is advisable to obtain good performance after seeding .
moreover , fungal contamination of seeds can lead to mycotoxins production such as aflatoxin b1 , b2 , g1 , g2 , m1 , ochratoxin a , zearalenone , and fumonisin .
it is thus common practice to treat seeds with antifungal molecules , but this leads to evident environmental problems and even to concern about safe disposal of seed packaging .
alternative solutions will likely become widely spread in the future and must be investigated . among them , ozone is wellknown for its efficiency as a surface disinfection agent , especially as it leaves no toxic residue once converted into oxygen .
stability of pure gaseous ozone is about 3 days at 20c and falls to about half an hour in practical conditions . as a potent oxidative agent , it is efficient against both gram or gram+ bacteria , fungi , viruses , protozoa , and also bacterial or fungal spores . apart from its use in water sanitation , it has also been proposed to lower pathogen microbial contamination on diverse plant seeds or bulbs ( triticum sp .
, brassica sp . , zea mays , hordeum sp . , phaseolus sp . , helianthus sp . ,  ) [ 1 , 4 , 5 ] .
a decisive advantage of ozone is its low toxicity on seed germs which allows to preserve excellent germination performance .
several methods can be used to assess ozone disinfection efficiency . apart from direct surface visual inspection
, one can also wash a seed lot and microscopically inspect the solution collected therefrom to identify foreign bodies ( sclerotes , spores ,  ) .
one can also incubate seeds over an ad hoc culture medium for a period of 5 to 7 days in suitable temperature and lighting conditions and observe colony development [ 6 , 7 ] .
we used such an approach to assess ozone surface disinfection performance on triticum aestivum ( common wheat ) seeds .
however it appeared to us that a more thorough scrutiny of the results was necessary . while colonies numbering did not point to a strong effect of ozonation
we thus used self - organizing maps ( soms ) to analyze surface colony area .
soms are a particular kind of artificial neural networks that learn to classify input vectors ( here pixel rgb intensity data ) according to how they are grouped in the input space .
they differ from competitive layers in that neighboring neurons in the self - organizing map learn to recognize neighboring sections of the input space .
thus , soms learn both the distribution ( as do competitive layers ) and topology of the input vectors they are trained on .
soms have been used successfully to perform segmentation of complex images , in particular from diverse food products such as cookies , fried chips , or potatoes [ 912 ] . in the present case ,
this allowed to discriminate quickly and easily between different fungi species while dodging the necessity to remove image background beforehand .
this approach showed a significant inhibition of fusarium growth after ozone treatment , while seeds retained a germinating capacity over 95% .
ozonation thus is qualified as an efficient treatment for seed surface disinfection , while som use allows to fine tune image analysis of fungi colonies .
a particular sample from cv louisart ( moisture 14 , 3% ) was selected for its known huge contamination by fusarium .  
ozone was produced by corona discharge using a benchtop generator ( c - lasky series ozone generator c - l010-dt , airtree technology europe co. , germany ) , with a maximal production of 2  g / h when fed with atmospheric air .
seed samples ( 60  g each ) were placed in a cylindrical vessel ( 15  cm in diameter ) .
they were exposed for a known time to ozone treatment by percolating gas flow ( 5  l / min , 0,8  mg
the vessel was shaken every minute during operation to ensure homogeneous contact between gas and grain surface .  
petri dishes filled with malt agar ( biokar diagnostics , france ) were used for fungi detection . for each assay
, a total of 300 seeds were settled in 30 petri dishes . in a particular petri dish
a seed was settled in the center , and nine others were settled equally spaced on the periphery ( figure 1 ) .
control samples were used , that is , untreated seeds and seeds commercially treated with celest ( fludioxonil , syngenta crop protection nv , belgium ) .
colonies were counted and inspected both macroscopically and microscopically to identify fungi species , as detailed in previously published methods [ 6 , 7 ] .
most displayed growth of a single fungal species and were counted one for that species .
petri dishes were then positioned over a sky blue background in a lighting cabinet ( gti minimatcher , gti graphic technology , newburgh , usa ) equipped with two 6500k daylight - balanced fluorescent lamps .
a digital camera ( nikon coolpix 4500 ) was used to take images ( figure 1 ) in jpeg format ( 640  480  pixels ) .
images ( 8  bit ) were then imported into matlab 2011 software ( the mathworks , inc . ,
three bidimensional soms ( hexagonal networks ) of 5  10 neurons each were created using the matlab neuron network toolbox .
the networks were limited to two dimensions only to match the flat shapes of the clusters of rgb values from the two fungi colonies types ( alternaria sp . and fusarium sp . ) and of the image background .
these soms were trained using three composite images created by sampling a representative set of regions from colonies of alternaria sp .
, from fusarium sp . colonies , or from the image background ( figure 2 ) .  
this allowed to account for the whole complex set of color shades present in the colonies . over training iterations ,
the som progressively unfolded to match the shapes and topologies of the color pixel distributions ( figure 3 ) .
the trained soms were then used to perform segmentation of images ( figure 4 ) . for each pixel presented to a som
, a single winning neuron would respond as being closest to these particular pixel rgb coordinates .
euclidean distance between the winning neuron and pixel coordinates was then computed and used to decide whether the considered pixel was part of an alternaria colony , of a fusarium colony , or of none of them ( thus being background ) . in either colonies ,
ratio of the number of pixels to the total pixel number of a petri dish would finally provide an estimation of colony surface percentage .
seed - germinative capacity was evaluated by testing 400 seeds for each treatment , placed into four essay boxes ( 100 seeds each ) .
essay boxes are 21  cm long , 12  cm broad , and 8  cm high , with a cover to guard against water evaporation .
this substratum is then disposed in the bottom of the boxes , with a constant height of 2  cm ( about 820  g per box ) . in each box ,
100 equally spaced small holes are perforated through the sand by way of a nailed plank ( nails protrudes 1  cm ) .
each hole then receives a seed , and covered boxes are first incubated for 7 days at 6 - 7c in a refrigerated cabinet .
they then stay for 8 days at 22c in a temperature - regulated cabinet lighted with 12 neon tubes ( philips tl - d58w/54 - 765 ) , with a cycle of 12 day hours and 12 night hours .

 triticum aestivum seeds , variety of louisart , were exposed to ozone treatment for different durations and then incubated over petri dishes for 7 days .
the only significant effect was a higher number of healthy seeds when treated with celest .
however , no significant effect could be seen for either alternaria or fusarium counts . a more thorough inspection of petri dishes however hinted at the existence of an effect of ozone treatment on colony surface . to test this , we measured colony surface by way of image analysis through a som methodology .
as shown in figure 6 , seed treatment with celest led to significantly smaller fusarium colonies ( about threefold ) ; no significant effect was observed on alternaria colonies .
seed treatment with ozone also led to significantly smaller fusarium colonies ( about threefold ) .
however , a significant effect was observed on alternaria colonies , with surfaces about twofold greater after treatment . 

figure 7 shows the linear negative correlation between the surfaces of fusarium and alternaria colonies , for seeds treated with ozone .
we see that reduced fusarium surface results in increased alternaria surface , with a correlation coefficient of 0.71 .
all seeds retained the same germinative capacity than control , that is , over 95% , with the exception of seeds treated with ozone for more than 10 minutes in our experimental conditions , where germinative capacity was lowered to 90% .
the use of som to operate segmentation of fungi colonies on petri dishes images proved to be very efficient , as was already the case in earlier studies [ 912 ] .
we were able to obtain fast and easy image treatment , with very good segmentation between fusarium sp . and alternaria sp . colonies .
whole colony surfaces were selected , due to the som taking into account the whole range of color shades for the two types of fungi colonies . as previously described [ 1 , 13 ] , we observed an effect of gaseous ozone treatment to reduce fungi seed contamination without germinative impairment , except for prolonged ozone treatment .
however , our som method to measure surface occupation by colonies allowed to obtain more precise and detailed data , which led to better understanding of treatment effects .
first , a simple count of colonies number showed no significant decrease except for celest control . by colony surface analysis
, we also observed a decrease of both fusarium and alternaria growth after celest treatment .
ozone treatment had a different effect , effective on fusarium only : it led to a decrease in fusarium surface occupation ( the number of fusarium colonies is not decreased , but colonies are smaller ) and to an increase in alternaria surface occupation .
thus , reduced growth of fusarium allows alternaria to occupy more surface on petri dishes .
alternaria could be less susceptible to ozone toxicity , due to a more efficient spore resistance .
also , alternaria could be settled deeper into seed tegument and thus less exposed to ozone attack .
from this , we conclude that ozone is a more selective disinfectant than celest , acting efficiently on fusarium .
enhanced growth of alternaria is attributed to lower competition after fusarium was killed by ozone treatment .
it would be of interest to use this method to obtain detailed data about seed disinfection for a greater variety of fungi contaminants .
there is still to test its limits in case of colonies presenting closely matching color shades .
 we submitted to ozone treatment triticum aestivum ( common wheat ) seeds severely contaminated by fungi . 
 fungi colonies developed when seeds were placed over malt agar medium in petri dishes ; fusarium sp . and alternaria sp . were identified . 
 however , conventional colonies counting did not allow a clear assessment of the effect of ozone disinfection . 
 we thus used self - organizing maps ( soms ) to perform an image analysis of colonies surface area that clearly showed a significant disinfection effect on fusarium sp . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions