Report MICOSAT F. Efficacy and Selectivity evaluation of Micosat F) (Crude inoculum + Bacteria and Saprophytic fungi)

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Report MICOSAT F Efficacy and Selectivity evaluation of Micosat F) (Crude inoculum + Bacteria and Saprophytic fungi) to increase the production on Potato in Sicilia Region (Italy) 1

TABLE OF CONTENTS 1. SUMMARY Page 3 2. EXPERIMENTAL CONDITIONS 2.1 Objective of the trial Page 3 2.2 GEP compliance Page 3 2.3 Site description Page 4 2.4 Trial conditions Page 5 2.5 Design and layout Page 6 2.6 Information on application Page 8 3 APPLICATION 3.1 Application Conditions Page 10 4. ASSESSMENTS 4.1 Mode of assessment, recording and measurement Page 11 4.2 Type, time and frequency of assessment Page 11 5. RESULTS AND CONCLUSION Page 21 2

1. SUMMARY The trial was carried out on Potato [Solanum tuberosum L. var. Universa], on field-grown, in Sourthern Italy (Sicilia), locality Augusta (SR), according to an experimental design with non-randomized blocks, side-byside, length-wise layout and 1 replications. They were compared three theses: 1. Untreated check: (UTK): fertilization background with an organo-mineral and granular NP distributed for sowing; 2. Treatment (TRT): fertilization background with organo-mineral and granular Micosat F at a dose of 30 kg / ha seed distributed. One month after the collection has been carried out with an application Micosat F Tab Plus, in fertirrigation, at a dose of 2 kg / ha. 3. Treatment NP (TRT-NP): background with organic-mineral fertilization and a granular NP together with the microgranular Micosat F, the latter always at a dose of 30 kg / ha sowing. One month after the harvest has been carried out with an application Micosat F Tab Plus, in fertirrigation, at a dose of 2 kg / ha. 2. EXPERIMENTAL CONDITIONS 2.1 Objective of the trial The primary objective of the trial was to determine the efficacy and selectivity of Micosat F, in Italy for increases in production on Potato against the Untreated check.. 2.2 GEP compliance The study was done according to the EPPO guidelines and Standard Operating Procedures POS of the Testing Facility: Guidelines Discipline Title Number PP 1/135 (3): General Standards Phytotoxicity assessment PP 1/152 (3) General Standards Design and analysis of efficacy evaluation trials PP 1/181 (3) General Standards Conduct and reporting of efficacy evaluation trials including good experimental practice The study described in this report was conducted in compliance with the principles of Good Experimental Practice (GEP) as defined by guideline 91/414/CEE of the European Union. The study Directorship was conducted by Giovanni Caprio, Director Testing Facility Biofarm srl, Via Mazzini, Vico VI n 1, 81047 Macerata Campania CE 3

2.3 Site description Trial Location: : Augusta Postal code: 96011 City (Town), County: Siracusa Region & Country:: Sicilia/Italia Geographic Latitude and longitude Latitude Nord GPS 37 14'15"36 N Longitude Est GPS 15 13'13"80 E Altitude 15 m a.s.l. 4

2.4 Trial conditions Tab.1 : Crop information Crop / Code Bayer Scientific Name Common Name / Variety Transplanting date 06.12.2012 Amount tubers for sowing 35 q / ha 1 / SOLTU Solanum tuberosum (L.) Patata/ Universa Growing and use-related characters Grading: Proportion of large tubers: high Susceptibility to diseases: Leaf blight: susceptible Tuber blight: susceptible to very susceptible. Common scab: fairly susceptible Virus X: susceptible. Virus A: susceptible. Virus Y: fairly susceptible Leaf roll: fairly susceptible Nematode RO1-4: resistant. Nematode PA2-3: susceptible Internal tuber defects: fairly susceptible to rust spots and lack spots slightly susceptible to hollow heart. Maturity: Medium-early Dormancy period: slightly short to fairly short Culinary quality: Consumption table use group A-B Dry matter content: very low Keeping quality: medium Growing system / Previous crops Single crop / Not relevant Planting space Distance between rows Distance in rows 0.18 m 2 /1plant 0.60 m 0.30 m Plant density/no of plants/ha (approx.) 55.000 (5.5 plant/ m 2 ) Plant arrangement Row Training system System of growing according to the traditional practice Irrigation system Drip irrigation Environment - Site type Field Date of harvest Third decade of May (25/05/2013) Soil condition Weed free Soil type - Texture Sandy - Loam % Organic matter 2,3 Fertility level Good Soil drainage Good ph soil 6.7 Crop conditions The crop was in good conditions at beginning of the trial. 5

2.5 Design and layout Tab.2: Experimental design Trial design Non-randomized blocks Number of variants 3 Replications 1 Plot length Plot width 34.2 m 10.2 m Plot size (Surface) 348.8 m 2 Rows / plot 17 rows Plants / row Plants / plot 114 1938 Distance between single rows 0,60 m Distance in rows 0,30 m Width x field 41,0 m Length x field 239,0 m Trial area Plots 3 Total cultivated area 9768 m 2 Soil application Plots 2 Treated area 8372 m 2 Foliar application No. of plants/ha (approx.) 55555 Plant density 5.55 plants/ m 2 Plants/trial 54264 Treated plants 46512 6

Tab.3: Layout of the plots Site Type: Field Trial Designe: no-racobl 01.01 02.01 03.01 TRT 1 TRT 2 TRT 3 UTK: Organic mineral fertilizer and Minerale fertilizer granular NP distributed seed TRT: Organic mineral fertilizer &Micosat F microgranular (30 Kg/ha) VS Micosat F Tab Plus WP (2 Kg/ha) TRT-NP: Organic mineral fertilizer & Organic mineral fertilizer & Micosat F microgranular (30 Kg/ha) VS Micosat F Tab Plus WP (2 Kg/ha) 1 2 3 7

2.6 Mode of application Table 4: Protocol Treatment - (Trial Schedule Treatment list) Efficacy and selectivity evaluation of MICOSAT F and MICOSAT F TAB PLUS for increases in production on Potato against the Untreated check.. Protocol ID: 12506912 Study Dir.:Giovanni Caprio Location: Prov. Siracusa Investigator.: Dr. Davide Ragusa/V. Esposito Trt Treatment Form. Form. Form. Product Product Product Product Growth Appl. Volume Volume No. Name Conc. Unit Type Rate* Rate Unit Rate Rate Unit stage Code Spray Unit 1 Untreated check - - - - - - - - - - - 2 MICOSAT F NEW Crude inoculum Biological component - 40 18,60 - % % MGR 30 KG/HA - 12.0 5,580 - KG s.a./ha KG s.a./ha 00 A - - MICOSAT F TAB PLUS WP Crude inoculum Biological component - 10 7,5 - % % WP 2 KG/HA - 200 150 - g s.a./ha g s.a./ha 42-44 B 1000 L/HA 3 With NP MICOSAT F NEW Crude inoculum Biological component - 40 18,60 - % % MGR 30 KG/HA - 12.0 5,580 - KG s.a./ha KG s.a./ha 00 A - - MICOSAT F TAB PLUS WP Crude inoculum Biological component - 10 7,5 - % % WP 2 KG/HA - 200 150 - g s.a./ha g s.a./ha 42-44 B 1000 L/HA Trt = Treatments (Tested theses) 8

1 UTK: Organic Nitrogen (N) 13% Fertilizer NP* 100 Kg/Ha 20 Kg/Ha 2 TRT: Organic Nitrogen (N) 13% Micosat F New Vs Micosat F TAB Plus WP 100 Kg/Ha - 30 Kg/Ha 2 Kg/Ha 3 TRT-NP: Organic Nitrogen (N) 13% Fertilizer NP* Micosat F New Vs Micosat F TAB Plus WP 100 Kg/Ha 20 Kg/Ha 30 Kg/Ha 2 Kg/Ha *Fertilizer NP (S) with Zinc (Zn) 8-28 (23) +2 Composition Total Nitrogen (N) 8% Ammoniac nitrogen 8% Phosphoric Anhydride (P2O5) 28% (water soluble in neutral citrate ammonium and water ) Anidride fosforica (P2O5) solubile in acqua Phosphoric Anhydride (P2O5) 17% (water soluble) Total Sulphuric Anhydride (SO3) 23% (water soluble) Total Zinc (Zn) 2% 9

3 APPLICATION 3.1 Application Condition Unit A B Application Date 10 DECEMBER 12 24 APRIL 13 Applicazion Interval Days T1 135 DAT1 Application Method (Type) Application for sowing Fertigation Application Time (Start/End) 08:30 / 10:30 09:00 / 11:00 Air Temperature (Start-End) C 10-14 17-19 Soil temperature C 8-10 12-14 Relative Humidity (Start-End) % 80-70 80-70 Soil conditions Umido Umido Wind speed Wind direction Rainfall immediately after application m/sec Days mm 0,8 (2,88 km/h) W (Zefiro) 5 2,5 1,4 (5,04 km/h) ESE (Euro) 12 0,8 10

4. ASSESSMENTS 4.1 Mode of assessment, recording and measurement The measurements were carried out on all plants in the plot, considering the number of tubers, the diameter and the yield per plot. 4.2 Type, time and frequency of assessment Tab.5: Average number of tubers per treatment Treatments Rate Kg/ha 17/04/2013 128 DAT1 0 DALA 13/05/2013 154 DAT1 19 DTA2 26 DALA 1 Untreated check - 3 5 2 TRT Without NP 3 TRT-NP With NP Micosat F VS Micosat F Tab Plus Micosat F VS Micosat F Tab Plus 30-2 30-2 4,5 6 4 8 Assessment of 17-04-2013 From a visual examination, the first assessment shows that the tubers on untreated check are less in number compared to the treated, are also of minor size, where as missing about a month (- 40 days) to the harvest, everything suggests that the tubers will continue to grow. The Treated has a greater number of tubers per plant (No.4,5), larger than the untreated check and uniform size. The Treated-NP has more tubers per plant (No. 4) compared with the Untreated check, all of uniform size, so the comparison is the best. 11

Fig 1:TRT (sx) - UTK (dx) Fig. 2: TRT_NP (sx) - UTK (dx) 12

Assessment of 13/05/2013 The results of assessments show: Untreated check (UTK): n 5 tubers per plant; Treatment (TRT): n 6 tubers per plant; Treatment NP (TRT-NP): n 8 tubers per plant. The Treated NP has all uniform tubers, the Treated has tubers greater size than the Treated NP and the Untreated check. Fig. 3 Untreated check (UTK) 13

Fig. 4 TRT 14

Fig. 5 TRT-NP Assessments on Harvest Harvest was made on 25-05-2013, the number of boxes per plot was counted the values obtained are showed and summarized in Table 7: 1. UTK were harvested n 64 boxes, the yield per plot was made of 1449.92 Kg, the production per hectare of about 420 q. 2. TRT: were harvested No. 76 boxes, the yield per plot was 1831.90 kg, the production per hectare of about 540 q. 3. TRT NP were harvested n 70 boxes, the yield per plot was 1856.75 Kg, the production per hectare of about 546 q.li. 15

Tab. 6: Yield per Plot Variants N Boxes The net yield (Kg) Increase % on UTK UTK 64 1.449,42 100 TRT 76 1.831,90 18,8% TRT-NP 70 1.856,75 15,6% Also were collected from plots compared, n 3 boxes, chosen among the most representative of the sample. The data obtained are summarized in the following tables and graphs. Tab. 7: Yield per plot by weight and as a percentage of different calibers Caliber (size mm) Variants Yield (Kg/Plot) 28/45 45/55 55/70 Large size > 70 Kg % Kg % Kg % Kg % UTK 1.449,42 334,40 23,06 127,36 8,8 988,16 68,15 - - TRT 1.831,90 88,54 4,83 559,34 30,53 1.150,20 62,79 33,82 1,85 TRT-NP 1.856,75 51,45 2,77 279,3 15,04 1.137,15 61,24 388,85 20,94 16

17

18

19

Tab. 8: Number of tubers and average weight per caliber (Ref. Boxes sample) Caliber UTK TRT TRT-NP (size mm) N of Tubers Average weight (g) N of Tubers Average weight (g) N of Tubers Average weight (g) 28 / 45 63 83 16 72,8 13 56,5 45 / 55 19 105 51 144,3 32 124,7 55 / 70 83 186 59 256,52 60 270,00 Large caliber > 70 - - 1 445,00 13 427,00 20

5 CONCLUSIONS In the plots with mycorrhizae there was a higher production compared to the untreated, in particular the 26 % more in the plot named "Treated" and 28 % more in the plot named " Treated NP ". The difference in production between the two plots with mycorrhizae was minimal, the 1,3% more in one named " Treated NP " In qualitative terms, however, the production obtained in the " Treated " was higher, in fact over 90% (see graphics ) consisting of tubers calibre between 45 and 70 mm (the most demanded by the market ). While in the "Treated NP " the effect of fertilization with granular NP was probably "exalted " by mycorrhization, infact about 21 % of the production consists of large size, tubers> 70 mm.finally in untreated check the higher incidence of tubers with caliber of 28 to 45 mm was found, which suggests that the basal fertilization was definitely integrated. Fig. 6: UTK 45 / 55 21

Fig. 7 UTK 55 / 65 22

Fig. 8 UTK 45 / 60 23

Fig. 9 TRT > 70 24

Fig. 10 TRT- NP 45 / 55 25

Fig. 11 TRT- NP > 65 26

The Untreated check was manifested presence of Phytophthora infestans, Alternaria solani and Rhizoctonia solani. This justifies the increases in production recorded in the treatments (thesis n. 2 and 3). Fig. 12: Harvesting and selection in box Fig. 13: Harvesting and selection in box 27

Fig. 14: Rhizoctonia solani Fig. 15: Rhizoctonia solani 28

Fig. 16: Infected plant by Alternaria solani 29

Fig. 17: Infected leaves by Phytophthora infestans 30

Fig. 18: Infected tuber by Phytophthora infestans 31