Seed-potato production through sprouts and field multiplication and cultivar performance in organic system

Organic farming requires the production or use of certified seeds from the same cultivation system and, the information of potato cultivars adapted to organic production system is important. The objective of this work was to study the feasibility of utilizing sprouts of different potato cultivars, seed multiplication and yield potential in field conditions without using agrochemicals. Three experiments were conducted: the first experiment using sprouts as seed under protected cultivation in Guarapuava, Paraná State, Brazil, from November 2011 to February 2012; the second experiment was field conducted in Candói, Paraná State, from May to September 2012 and the third experiment was field conducted in Guarapuava, Paraná State, from February to May 2013. The experimental design was a randomized block design with 3 replications in all 3 experiments. In the 2 first experiments the following cultivars were evaluated: BRS Ana, BRS Clara, BRS Eliza, Crystal and Catucha, and in the last experiment Agata, Bintje, Baronesa and BRSIPR Bel were included. The traits analyzed were length of main stem, number of stems, number of leaves, fresh weight and number of tubers, percentage of infection by viruses and in the second experiment the incidence of late blight on leaves. In the third experiment, leaf area index (LAI), tuber, leaves and stem dry weights and late blight severity were also analyzed. In the first experiment, the cultivars showed differences in plant height and minituber production, the highest value was observed in cultivars BRS Clara and BRS Ana for plant height and cultivar BRS Clara for minituber production. In the second experiment, there was difference only in plant height, the tallest cultivars were BRS Ana and BRS Clara. There was low spread of viruses and, it did not directly reflect in tuber yield. In the third experiment the cultivars with lower disease severity were those recommended to organic farming, especially BRS Clara and Catucha; this second cultivar had the higher LAI and tuber yield, as well as resistance to late blight, factors that might have contributed to the recorded highest total tuber yield (13.8 t/ha).

of agrochemical and fertilizers.It is well known that a major constrain on organic potato production is disease with great destructive potential, such as late blight (Phytophthora infestans).Therefore, the correct use of late blight resistant cultivars is fundamental for satisfactory yields on organic cropping system (Möller & Reents, 2007).
In 2013 the Brazilian legislation for organic cropping systems imposed that seed or propagating material, should be originated from organic systems, for all organic crops (MAPA, 2008).Therefore, research for suitable and feasible organic seed-potato production systems has been demanded.Potato seed production under organic system is complex due to the high incidence of pathogens causing seed-potato degeneration, particularly viruses, like Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV).Like most potato viruses, these two are seed-tuber borne, resulting in expressive yield reduction and table-quality depreciation (Rahman et al., 2010).
Searching for alternative organiccertified seed-potato production systems, the sprout/seed-potato technology has been considered as a sustainable, low cost system for large scale production of seed-potato stocks (Silva et al., 2006;Souza-Dias et al., 2011).However, the sprout/seed-potato technology has not yet been tested in Brazil for organic seed-potato production, nor, either for field multiplication of seed-potato cultivars on organic production system.
The present work was carried out aiming to evaluate: 1) the multiplication potential and sanitary status of seedpotato stocks from different potato cultivars, produced from sprouts as original propagating material, without use of agrochemicals in protected and in field cultivation systems; and 2) its successive growth and yield of the different potato cultivars originated from the organically produced seedpotato stocks.

MATERIAL AND METHODS
The experimental work was split in three phases:

Summer sprout cultivation under insect-proof greenhouse
The experiments were carried out inside an insect-proof greenhouse at Feroz Farm, located in the region of Guarapuava (25°26 S, 51°34 W, altitude 1016 m), from November, 2011 through January, 2012.
Seed-potato tuber lots from five different potato cultivars classified as basic were supplied by Embrapa-Canoinhas Products and Marketing, whith indication as most fitted for organic cropping: BRS Ana, BRS Clara, BRS Eliza, Catucha and Cristal.All cultivar's seed-potato tuber lots were certified with zero percentage of Potato Leafroll Virus (PLRV) as well as Potato Virus Y (PVY).The experimental design was random blocks of 12 pots with 3 replications of each cultivar, totalizing 180 plastic pots containing 1.5 L of a commercial soil substrate, composed of processed pine and coconut fibers, plus expanded vermiculite and turf.
On November 13 th 2011, apical single sprouts measuring 5-10 cm high were detached from the tubers, placed in zipped plastic bags, stored in a refrigerator overnight and planted, one per pot.Sprouts were vertically inserted at 2 primordial bud nodes deep in previously wetted substrate and at least one bud node was left outside.
At 25 and 45 days after planting (DAP), number of emerged stems, formed leaves and plant height were recorded in 6 plots per treatment.Despite of the maturity stage, using a razor blade the stems of each plant were cut at base in order to standardize the harvesting, which took place at 73 DAP, when records were taken on the diameter, fresh weight and amount of tuber (minituber) produced per sprout.Immunoassays (DAS-ELISA) test on the sprout of 15 minitubers, representing one plant each, were performed in order to evaluate the presence of PLRV and PVY in each cultivar.

Winter field planting
The second experiment, using as seed-potato the minitubers produced on the previous experiment, took place at Itaguaçu Farm, in the region of Candói (25°44 S, 52°13 W; altitude 559 m), during the months from May through September, 2012.
Each field experimental plot was represented by 36 plants distributed in 4 rows, 2.25 m long, 0.75 m between rows and 0.25 m between plants.The plots were randomly distributed with 3 replicates of the 5 same cultivars planted in the first experiment.On the 5 th May, 2012, minitubers were planted by hand without any chemical treatment, throughout the experiment.
Prior to potato, the experimental area was planted with corn, also cultivated without chemical fertilizer nor any chemical treatment.Field soil preparation took place 25 days before planting with a single subsoiler and rotary tillage procedures.For soil fertilization, we applied at once 1000 kg/ha high calcium (calcitic) lime (CaO: 50% and MgO: 2%, PRNT= 90%) and 1000 kg/ha of reactive natural phosphate (P 2 O 5 : 29%).
The emergence was evaluated at 15 and 25 DAP, when at least 70% of the plants started breaking up above the soil.At 45 and 65 DAP the number of emerged stems, expanded leaves and plant height were measured from the 6 plants at the central rows.The incidence of late blight natural infection was recorded on foliage at 65 DAP.Harvesting took place when plants reached natural senesce or physiological maturity stage, being recorded tuber diameter, fresh weight, quantity and total yield of 10 plants in the two central rows, excluding the 2 plants at the beginning and at the end of each row.
To determine the occurrence of virus spread and the incidence of the two major seed-potato viruses (PLRV and PVY), one tuber per plant/cultivar was taken and sprout/DAS-ELISA tested.
The experiments were arranged in a randomized block design, with 9 treatments and 3 replications.Each block consisted of 64 seed-potato tubers distributed in 8 rows spacing 0.8 m wide between furrows and 0.25 m between tubers.The treatments consisted of the following potato cultivars: Agata, Baronesa, Bintje, BRS Ana, BRS Clara, BRS Eliza, BRSIPR Bel, Catucha and Cristal, obtained from local seed-potato producers or Embrapa Canoinhas Products and Market.
Following a deep moldboard plow and conventional potato soil tillage, involving a disc plowing, raised seed beds were prepared.At planting time, furrows fertilization (mixed with the soil) was applied with 10 m³/ha of sheep manure; 10 m³/ha of poultry manure and 1000 kg/ha of natural reactive phosphate.On February 5 th , 2013, hand-planting (0.10 m deep) was carried out while counting of above ground (emerged) plants took place at 26 DAP.Ridges were mechanically made at 38 DAP and at the 40 th DAP a 1% (v/v) Bordo mix was sprayed at 200 L/ha of solution.
Natural occurrence, disease progress and symptoms severity of late blight were evaluated 4 times, from the 55 th until the 76 th DAP, on a 7 days interval.According to James (1971), records were made, for each cultivar, on the basis of total percentage of foliar area damaged, where a rate of 100% damage was recorded for completely plant death, due to the disease; while 0% was recorded for none visual symptoms on total foliar area of a plot.Following records taken to late blight severity, we calculated the area under disease progress curve (AUDPC) over the same period considered (55-76 DAP).
Plant height and leaf number of 6 plants per plot were evaluated at 45 and 72 DAP.At 72 DAP, on a destructive harvest of 4 plants per plot, the following evaluations were performed: leaf area index (LAI); total accumulated dry weight distributed among leaves, stems and tubers; as well as fresh weight and tuber numbers.To determine dry weight, the samples were placed into a forced air circulating chamber at 70°C until constant weight was reached.The LAI was estimated by means of quantification of specific foliar area, for each plant, with digital photos taken from leaf sub-samples and compared against standard area of 5x5 cm, using the ImageJ 1.41 software (Abramoff et al., 2004).
On May 23 rd , of 2013, at 107 DAP, final harvesting was performed for 10 plants per plot, considering only the 6 central rows, not considering the first plants of each row.The total yield was estimated from the number of tubers and their total fresh weight, while the commercial yield was estimated on the basis of yield of tubers sizing over 45 mm of diameter.In this experiment, viruses incidence was not evaluated.
All data were submitted to analysis of variance and the means were separated by Tukey test at 5%, using the software package Assistat version 7.6 beta (2012).

Summer protective cultivation of sprouts
Under protective cultivation, the development of the primary leaflets was similar among all different potato cultivars, as recorded at 3 DAP.This performance could be explained for the fact that sprouts, as propagating material, are naturally capable of fast leaf differentiation (Daniels-Lake & Prange, 2007).In addition, this differentiation could have been facilitated by the fact that each sprout was planted with at least one axial node above soil.No difference among number of shoots (stem) was observed among cultivars, while an average 1.17 stems per sprout was recorded from 25 until 45 DAP.Kawakami et al. (2003) also observed only one stem grown from propagating material of small size, such as microtubers weighting 1.0 g.These results suggest that small size propagating material, such as sprouts or microtubers, will generally emerge a single-stem plant.
No significant differences were recorded on developed leaves among cultivars; an experimental average of 7.0 leaves were recorded at 25 DAP, while 8.7 leaves at 45 DAP (Table 1).A difference on plant height among cultivars was recorded: BRS Ana and BRS Clara showed the highest plants on both first and second evaluations; while cultivar Cristal revealed shortest plants at both evaluations.Virmond et al. (2011) observed average of 9.5 cm tall, at 20 DAP, for cultivar Agata plants originated from sprouts, using same type of soil substrate, with mineral fertilizer.These results corroborate with those from the present study.
The plants from the different cultivars originated from sprouts produced similar tuber size and quantity, however with differences on fresh weight, what result in differences per pot production.The average diameter of tubers was 3.7 cm, a result close but lightly superior from those from Factor et al. (2007): 3.0 cm, recorded for sprouts planted in a hydroponic system.On the present work, an average of 3.0 tubers were produced per plant, an amount inferior of that observed by Silva et al. (2006): 4.4 tubers per plant grown on different substrate soil mix under protected cultivation system.Virmond et al. (2011) reported, also under protected environment, an average of 4.0 tubers per plant from sprout on a single substrate soil, although fertilized with granulated NPK before planting.Queiroz et al. (2013) observed that the increase on tuber number/area, is a function directly associated with increase on fertilization, which would explain, in part, the smaller number of tubers observed in the present work as compared to those observed from Silva et al. (2006) and Virmond et al. (2011), as no mineral fertilizer was Seed-potato production through sprouts and field multiplication and cultivar performance in organic system applied in the present studies.Plants of cultivar BRS Ana produced minitubers containing higher fresh weight (18.3 g) than plants of cultivars BRS Eliza, Catucha and Cristal (average of 11.8 g).The average fresh weight of these minitubers were higher than those reported for cultivar Agata, by Silva et al. (2006) and Virmond et al. (2011): 4.5 and 4.0 g, respectively.It is possible that the more minitubers produced by theses authors were inversely related to the less fresh weight as reported in their experiments.
Only one tuber of cultivar BRS Eliza was infected with PVY, representing 6.67% of the total analyzed tubers.This result sustains previous observations that using sprouts as propagating material under protected cultivation can result in high grade, low virus incidence (near virus-free) minitubers/seed-potato stocks.

Winter field cultivation
For all cultivars, over 70% of plants had emerged at 25 DAP and, in average 85% of plants at 45 DAP, from the total tubers planted.Kawakami et al. (2003) also recorded a high percentage (97%) of plant emergence from microtubers weighing 1.0 to 3.0 g, revealing that even small tubers can result in near 100% emergence.
Plant height from tubers of different cultivars varied, although maintaining similar foliage number (Table 1).Readings taken at 45 and 65 DAP showed cultivars BRS Ana and BRS Clara with plants higher than cultivar Cristal.The measurements recorded in the present studies were lower than those recorded by Queiroz et al. (2013), whose records for cultivar Agata at 41 days after emergence in field cultivation on summer season were 46.0 cm in average, without addition of fertilizer.This difference probably may be explained because the present study was carried out in winter season with average temperatures lower than those recorded by Queiroz et al. (2013) in summer season.In addition, the higher size of pot plants experiments, as recorded for cultivars BRS Ana and BRS Clara, would agree with the field experiments, corroborating with evidences that these two cultivars are characterized by high plants.The average number of foliage for each cultivar at 45 and 65 DAP was 6.0 and 8.7, respectively.These values are below the 18 leaves recorded in same season experiments by Bisognin et al. (2008), working with different potato clones, cultivated under conventional system, including mineral fertilization.
Evaluations at 65 DAP showed a significant difference on natural occurrence of late blight among all potato cultivars.An incidence of 36% for plants of cultivar BRS Ana, 22% for cultivar BRS Clara, 5% for cultivar BRS Eliza and 2% for cultivars Catucha and Cristal were observed, showing differences on initial infection resistance among the cultivars.Möller & Reents (2007), in Germany, also observed variable resistance level in 8 potato cultivars grown on eight years of evaluation under organic cultivation system.
The cultivar BRS Ana, due to a severe damage caused by late blight, was harvested at 90 DAP, while the other cultivars were harvested when the cycle (natural senescence) was completed: cultivar Cristal, at 110 DAP, the others at 140 DAP.No significant difference was recorded among treatments in none of the cultivars analyzed.The tuber diameter recorded in these experiments averaged 4.1 cm which would be fitting at class type II on the certified tuber/seed-potato classification.Regarding tuber diameter as a function of yield, Queiroz et al. (2013) observed that 4.1 cm seed-potato size did not differ in comparison with seed-potato of larger size.Since tuber/seed-potato lots are commercialized on the basis of fresh weight, this results could turn out to be a good point as economical option to growers.
The tubers produced in the present work showed an average size of 23.0 g.Based on Kawakami et al. (2003) and Kawakami & Iwana (2012), as long as cultivation is carried out in ideal conditions, this average tuber fresh weight can be considered as satisfactory as these authors demonstrated that tuber/ seed-potato weighing less than 1.0 g, i.e., 20 fold lighter in weight than tubers produced in the present work, did not reveal major differences in yield, when compared with plants originated from conventional tuber seed weighting around 50 g.
The average number of tubers per plant was 2.3, which is considered low if compared with the average of 7.0 tubers, as observed by Kawakami & Iwana (2012), using microtubers/seedpotato of an average of shorter size than the tubers used in this system, however, using mineral fertilization and cultivars of longer cycle.The low number of tubers can be explained by the fact that a drought season, lasting 42 days was recorded, with a water deficiency that is known to cause potato plants to produce less tuber number as well as fresh weight (Hassanpanah, 2010).Giusto (2006) observed an average potato tuber production of 13.9 t/ha under conventional field cultivation in Alfenas, Minas Gerais State (Southeast of Brazil), using minituber/seed-potato originated from sprouts, on a controlled environment cultivation during fall season in Campinas, São Paulo State.In the present studies, the average yield of 4.1 t/ha could be due to the fact that cultivation seasons and geographic locations were very different: winter in the south of Brazil, when generally the solar radiation and temperatures are lower, as well as precipitation.Also, in the present studies, a hail precipitation was observed on June 7 th , 2012, at 33 DAP.Temperature recorded 0.8°C for minimum, a fact that could also had been contributed to reduced tuber number and yield.Indeed, Zarzynska & Pietraszko (2015) concluded that potato cultivated on organic systems is more severely impacted by climatic variations than potato cultivated under conventional systems.
Concerning viruses infection, the same percent occurrence of virus infection by PVY in tubers of cultivar BRS Eliza (6.7%) in the first experiment, was also recorded in one plant of cultivar Catucha, probably due to aphid transmission within the experiment.

Summer field cultivation
The plant emergence, at 26 DAP, was similar among the treatments, showing an average of 86%.
Considering late blight severity evaluations, cultivars BRS Clara, Catucha and Cristal showed the highest resistance at 76 DAP, when last records were taken.Such result was observed associated to a low AUDPC as shown by these cultivars (Figure 1).Cultivar BRS Clara was the most tolerant as none disease symptoms were recorded until the last evaluation.These results sustain Pereira et al. (2013) whose records for late blight concluded that BRS Clara possess foliar resistance against late blight.On the other hand, cultivar Agata showed a high susceptibility to late blight, as a high AUDPC record was recorded on this cultivar: at the 55 DAP, plants of cultivar Agata showed no effective photosynthetic leaf area due to severe disease incidence.For cultivar Bintje, the total loss of photosynthetic leaf area was recorded at 69 DAP, standing as a second most susceptible cultivar, with high AUDPC.This results corroborate Töfoli et al. (2013) on their observations that cultivars Agata and Bintje were susceptible to late blight.In addition, it is known that potato cultivars with short cycle are more susceptible to late blight than long cycle ones (Brune et al., 1994), a result that was also observed in this study on cultivars Agata and Bintje, the two most short cycle cultivars.The high late blight severity as recorded in the susceptible cultivars could have been a response to the climatic records: 17°C, with distributed precipitations maintaining foliage wet for an ideal disease progress during the whole cycle (Töfoli et al., 2013).
On the first evaluation, taken at 45 DAP, there were no significant differences in plant height among the studied cultivars (Figure 2).On the second evaluation (72 DAP) a difference among cultivars was recorded due to a complete late blight destruction of photosynthetic leaf area on plants of cultivar Agata.The recorded values were lower than those from Queiroz et al. (2013) whose records showed an average of 46 cm on cultivar Agata, at 57 days after emergence, without applying mineral fertilizers.
Regarding the number of developed leaves, records taken at 45 and 72 DAP showed distinct groups, with one of them striking for its most foliage counting: cultivars Catucha and BRSIPR Bel; and the other group formed by cultivars Agata, BRS Ana and BRS Eliza, showing the least foliage counting.The remaining cultivars showed an Potato cultivars grown under organic system at summer field crop: plant height and number of leaves per plant at 45 and 72 days after planting; tuber, stem, leaf and total dry weight at 72 days after planting.Guarapuava, IAPAR, 2013.ns 1 : not significant difference (p>0.05); 2 averages followed by same lowercase letter do not differ according to Tukey test, 5%; 3 averages followed by same uppercase letter do not differ according to Tukey test, 5%.

Figure 1 .
Figure 1.Area under disease progress curve (AUDPC) of potato cultivars affected by late blight (Phytophthora infestans) under organic cultivation at summer field; ( 1 averages followed by the same letter do not differ according to Tukey test, 5%).Guarapuava, IAPAR, 2013.

Figure 2 .
Figure2.Potato cultivars grown under organic system at summer field crop: plant height and number of leaves per plant at 45 and 72 days after planting; tuber, stem, leaf and total dry weight at 72 days after planting.Guarapuava, IAPAR, 2013.ns 1 : not significant difference (p>0.05); 2 averages followed by same lowercase letter do not differ according to Tukey test, 5%; 3 averages followed by same uppercase letter do not differ according to Tukey test, 5%.

Table 1 .
Potato production per pot and yield of tubers grown in greenhouse and in field under organic system: number of leaves, plant height at 25, 45 and 65 days after planting (DAP) and tuber diameter, fresh weight and number per pot and per plant.Guarapuava and Candói, UNICENTRO, 2011/12.Averages followed by same letter in columns, do not differ according to Tukey test, 5%.

Table 2 .
Field grown potato plants at summer, under organic production system: fresh weight, number of tubers, leaf area index (LAI) at 72 days after planting (DAP) and tuber yield for total and commercial number of tubers.Guarapuava, IAPAR, 2013.Averages followed by same letter in columns, do not differ according to Tukey test, 5%; --= plants without leaf area due to late blight (P.infestans) infestation.