Objective classification of Penicillium fungi. May 22, 2006

Transcription

1 Le H. Objective classification of Penicillium fungi Le H. IMM Michael E. Hansen and Bjarne K. Ersbøll May 22, 2006 Pol YES

2 Overview Le H

3 Background Le H.

4 We live a fungal world Le H. Fungi are the second most diverse organism on earth - after sects.

5 We live a fungal world Le H. Why study fungi?

6 We live a fungal world Le H. Estimatg the number of fungal species [Hawksworth, 2001] 1 : Flowerg plants on British Isles described = 2,000 species Fungi on British Isles described = 12,000 species Ratio of 6:1 fungi to each plant species Total number of described plant species = 250,000 (most plant species are believed to be described) 6 x 250,000 = 1.5 million species of fungi! Less than 5% of which are described, and at the current rate of description, it will take > 800 years to describe all extant species. Mycologists advertently redescribe already known species at the rate of about 2.5:1. [Hawksworth, 1991] 2 1 Hawksworth, Myk. Res., 105, 2001, p Hawksworth, Myk. Res., 95, 1991, p

7 The Penicillium genus Le H. The filamentous fungal genus Penicillium is one of the most important fungal genera. Different species: Produce important drugs, e.g. penicill and compact. Are used food fermentation, e.g. white cheeses, P. camemberti; blue cheeses, P. roqueforti and mold fermented salami, P. nalgiovense. Deteriorate foods and other materials.

8 to species level Le H. is: Difficult due to mute differences appearance: Conidium (spore) colors, diffusible pigments, exudates, droplets and texture. Subjective. Three species 3 are subject to classification. For each specie four isolates are regarded. Mel YES Pol YES Ven YES 3 From the IBT culture collection held at BioCentrum, DTU.

9 Le H. Disadvantages usg several media: Expensive Time consumg Methods to choose one or two media are desired. Three media are tested for their formative level to the identification: Czapek yeast extract agar (CYA) Oatmeal agar (OAT) Yeast extract sucrose agar (YES)

10 - Videometer Lab Le H. Wavelength 430nm 450nm 470nm 505nm 565nm 590nm 630nm 645nm 660nm 700nm 850nm 870nm 890nm 910nm 920nm 940nm 950nm 970nm Color Ultra blue Blue Cyan Cyan Amber Orange Red Red Red Red NIR NIR NIR NIR NIR NIR NIR NIR

11 analysis - Segmentation Le H. ROIs the images have been segmented usg a method called Histogram Pursuit. The method looks for nonlear distributions of n classes. Example:

12 analysis - Features Le H. s: Origal Features: Pairwise differences between spectral bands Pairwise multiplications between spectral bands Mean, standard deviation, and maximal tensity 1st, 5th, 10th, 30th, 50th, 70th, 90th, 95th, and 99th percent fractiles

13 Multivariate analysis of variance Le H. MANOVA is performed to check the: Validity Reproducibility Cross classifications and hierarchical classifications the expermtal design: S M S I I M S R I R M R.

14 Multivariate analysis of variance - Models Le H. X kljν = µ+ m k + s l + ms kl + i(s) j(l) + mi(s) kj(l) + and R(msi) ν(klj)(1) X kljν = µ+ m k + s l + ms kl + I(s) j(l) + mi(s) kj(l) + R(msI) ν(klj), (2) where X kljν is an observation of the two first PCs with k = 1,...,3 l = 1,...,3 i = 1,...,4 ν = 1,..., 3 (medium) (specie) (isolate with specie) (repetition with medium, specie and isolate)

15 Multivariate analysis of variance - Results Le H. The two first prcipal components are used for the analyses. Null-hypotheses that variances or terms of effects are zero are tested at a 5% level of significance. Determistic model: All effects and their teractions significantly different from zero. Mixed model: The variance of the isolates not significantly different from zero.

16 Statistical tests for choice of Le H. The statistical tests: Hotellg s T 2 test: H 0 : µ 1 = µ 2 Test of additional formation: H 0 : Last q variables do not contribute to the discrimation are used to determe which medium and/or combations of media are best with respect to discrimation between species.

17 - Results Le H. Tests: Choice: All mean values are significantly different Mahalanobis distances are largest on the YES medium 2 media compared to 3: H 0 is accepted 1 medium compared to 2: H 0 is rejected except for YES & OAT 1 medium: YES 2 media: YES & CYA

18 Discrimant analysis Le H. Bayes rule: Choose the population where the observed probability is largest, i.e. the population a given observation is more likely to be observed. Lear functions are used to discrimate between the populations. This means that we assume that the three populations have equal dispersion. We have: 36 observations and approx features. Hence, feature selection is a necessity. Forward selection based on Wilk s Λ is performed. Leave-one-out cross-validation and test set validation has been used.

19 Discrimant analysis - Results Le H. 100% correct classification usg 2 variables on YES. DA Mel Pol Ven DA2 DA Mel Pol Ven DA2 Var DA1 DA2 DA3 Difference Difference Difference Parameter 99th percentile 30th percentile 5th percentile Bands cyan & amber ultra blue & red ultra blue & NIR (nm) (505&590) (430&645) (430&870)

20 Le H. The is reproducible with same and with other isolates. A method to choose the best has been proprosed. One medium (YES) is sufficient practice, whereas two media (YES and CYA) theory are the best choice. The classification is 100% correct usg both leave-one-out cross-validation and test set validation. Two variables is sufficient for the classification.

21 Future Le H. A larger study with several species and several media to verify the method for choosg the best media. Usg the chosen media for classification, perhaps for another genus, e.g. Aspergillus.