Improving the Genetics of Tuber Yield and Resistance to Mite to Avoid Mite Incident and to Increase the Productivity of Cassava (Manihot esculenta Crantz)

Mite (Tetranychus urticae) that attacks the cassava plants during dry season can reduce the yield up to 53%, depending on plant age and duration of attacks. The objective of the trial was to evaluate the cassava promising clones for tuber root yield and mite resistance. The field trial was done in Malang, East Java, Indonesia, in 2018 with fifteen clones using a randomized complete block design, with three replications. The glass house experiment for mite evaluation was done in Malang in 2018. A total of fifteen clones were used in this glass house trial. Infestation of mite (imago) was done 1 month after plating with 15 mites/pot on the lower part of the fourth or fifth leaf. Results of the field experiment showed that there was a genetic variability in the clones tested. The fresh tuber yield in 10 months ranged 30.33–55.67 t/ha with mean 41.34 t/ha. The fresh tuber yield of clone OMM 0915-11 was the highest of 55.67 t/ha. The tuber result of clone UJ5d50-207-3 similar to OMM 0915-11 and significantly higher than check variety UJ5. Response of clones to mite attack were as follows: two clones were resistant, ten clones were moderately resistant, and the other clones were susceptible. Based on the green house trial, the response of clones to mite attack was as follows: one clone was highly resistant, two clones were resistant, ten clones were moderately resistant, and the other clones were susceptible. Clone OMM 0915-11 was resistant variety or high resistant variety based on the green house and field experiments, while clone UJ5d50-207-3 was moderately resistant based on both glass house and field experiments.


Introduction
Productivity of cassava is important characteristic related to farmer income per crop area and harvesting time. Productivity of cassava is determined by genetic factor and environment factor. Many released varieties can be used by farmers. Air temperature, sun light, and rain fall are the environment factors that can not be modified, while fertilizer (dosage and kind), planting distance, and irrigation are the environment factors that can be modified.
Fertilization is one of the environmental factors that can be modified to increase productivity of cassava. Kind and dosage of fertilizer needed to get the high productivity depended on the soil fertility and the kind of variety used. Senkero et al. [1] reported that fertilization 0-80 kg·N/ha gave the significantly positive response to yield in all locations of the experiments with characteristic as follows: N content in the soil 0.5-1.4 g/kg, K 0.15-0.81 cmolc/kg, and P 7.6-16 mg/kg, while fertilization with 0-22.5 kg·P/ha and dan K 0-30 kg/ha did not give significant response to yield. e cassava productivity is also affected by insect severity that attacks the crops in the field. An important insect in cassava is mite (Tetranychus urticae) [2]. In general, this insect attacks the plant during dry season. Mite can reduce the yield 20-53%, depending on plant age and duration of attacks. Indiati [3] reported the loss of yield of 8-54%.
Ezenwaka et al. [4] reported that the loss of yield of cassava due to green mite (Mononychellus tanajoa (Bondar)) was over 80%. To handle the mite attack, the farmer uses resistance variety. Use of resistance variety is easy and cheap and causes no pollution for the environment. So, new varieties should be moderately resistant or resistant to mite. e role of plant breeding is important in increasing the productivity and the mite resistance of cassava variety. Conventional breeding is still dominant in producing new varieties. Germplasm evaluation is first step in conventional breeding, followed by clonal selection. Clonal selection involves single plant selection, single row selection, and single plot selection. After that, they are followed by preliminary yield trial and advanced yield trial [5].
ere were some promising clones from breeding activity that started in 2009. Some promising clones resulted from hybridization and some others resulted from mutation using gamma irradiations. e objective of the trial was to evaluate the cassava promising clones for tuber root yield and to determine the mite resistance.

Study Sites.
e study consisted of field and glass house experiments which were conducted in Malang, East Java, Indonesia, in 2018. e field experiment was conducted in the Jambegede Experiment Farm which is located at the coordinate of −8°10′20″ South Latitude and 112°33′43″ East Longitude, with 335 m above sea level. e glass house experiment was conducted in Kendalpayak which is located at the coordinate of −8°2′51″ South Latitude and 112°37′30″ East Longitude, with the altitude of 436 m above sea level.

Field Experiment.
e field experiment was done using a randomized complete block design, with three replications. A total of fifteen clones were tested in this experiment. e plot size was a 5 m × 4.8 m and plant distance was a 100 cm × 80 cm. Plants were fertilized three times, first fertilization was done in 2 weeks after planting 15 grams of phonska (N 15%, P 15%, K 15%)/plants, second fertilization was done in six weeks after planting 20 grams of phonska (N 15%, P 15%, K 15%)/plant, and third fertilization was done in 10 weeks after planting 20 grams of phonska (N 15%, P 15%, K 15%)/plant.
Cassava stake with a 25 cm long was planted in a pot (1 plant/pot). Diameter of pot was 30 cm with 5 kg soil/pot. Fertilization was 5 grams of phonska (N 15%, P 15%, K 15. Irrigation was given based on the needs. Infestation of mite (imago) was done in 1 month after planting with 15 mites/ pot on the lower part of the fourth or fifth leaf.

Data Collection
e variables taken were the fresh tuber yield, starch content, and plant height in seven months, number of tubers/plant, diameter of tuber, harvest index, and mite attack with natural infestation. Starch content was taken based on gravity system with wet basis. Starch content was obtained from multiplication between specific gravity and 112.1 minus 106.4. Specific gravity was gotten from the fresh tuber weight in the air divided by the value of the fresh tuber in the air minus the fresh tuber in the water.
e damage intensity of the mite attacks was calculated as follows: where I is the damage intensity of the mite attack, N is the number of leaves/plant, V is the highest score (5), nis the number of leaves in each score category, and v is category score (from 0 to 5). Scoring was done as follows: 0 � no symptoms; 1 � Initiation of yellowish spots on some of the lower and/or middle leaves; 2 � fairly abundant yellowish spots on lower and/or middle leaves; 3 � considerable damage: many spots; small necrotic zones and curling of leaves, especially the basal and middle leaves; yellowing and loss of some leaves; 4 � severe damage: heavy defoliation in the lower and middle part of the plant; a large number of mites as well as webs can be observed; 5 � total defoliation of the plant; shoot reduced in size with large number of webs; death of plant.
Susceptibility class was determined using the Chiang and Talekar [6] formulas as follows: Highly resistant(HR) � I <(I − 2δ), where (I) is mean of the damage intensity of the mite attack. δ is standard deviation.

Glass House Experiment.
Data were taken on mite population during 3-9 weeks after planting, the damage intensity of mite attack during 3-9 weeks after planting, color of leaf in 3 months after planting, and pubescent of leaf in 3 months after planting.

Data Analysis.
e damage intensity of the mite attacks and susceptibility class was calculated using a MSTAT-C software to analyze the data and Minitab software was used to develop the dendogram. Mite attacks intensity in 10 months of plant age ranged between 24.14% and 51.32% (Table 2). Clone OMM 0915-11 had the lowest intensity of mite attacks (24.14%), which was resistant variety. Clone OMM 0940-2 was also resistant variety. Clones UJ5d50-99-1 and UJ5d50-207-3 were moderately resistant variety, while UJ5 was susceptible variety ( Table 2). Harvest index of tested clones was in the range of 0.48-0.65. Clone CM 3306-19 had the highest harvest index (0.65). Clone UJ5d50-99-1 had harvest index similar to CM 3306-19 and significantly higher than UJ5. e average tuber (≤100 gram) diameter ranged from 4.36 to 5.87 cm. Variety UJ5 had the biggest diameter (5.87 cm). e tuber of clone OMM 0915-11 was similar to UJ5. e number of tubers (≤100 gram) ranged from 9 to 17 tubers/ plant; however, this difference was not significant statistically. Tuber length of tested clones ranged from 20 to 29 cm; however, this difference was not significant statistically.

e Field Experiments
Dendogram analysis of 15 cassava clones based on plant height in three months, fresh tuber yield in 10 months, starch content, starch yield, intensity of mite attack, harvest index, tuber diameter, and number of tubers/plant is presented in Figure 1. If cutting is done with similarity of 40.47%, three clusters will be obtained (Table 3). ere are 11 cassava clones (2; 14; 5; 3; 10; 13; 12; 11; 7; 8; 9) in the first cluster. e characteristics of these clones were low/medium tuber yield, low/medium starch yield, high/medium starch content, low/medium/high plant height, and susceptible/ moderately resistant/resistant to mite. ere are two clones (1 and 15) in second cluster. e characteristics of these clones were high tuber yield and starch yield, high starch content, low/medium plant height, and moderate resistance/ resistance to mite. ere are two clones (4; 6) in the third cluster. e characteristics of these clones were high tuber yield and starch yield, medium starch content, medium plant height, and moderate resistance/resistance to mite. Figure 2 shows that mite population had increased in 3-4 weeks after infestation, and in 5 weeks after infestation, mite population had decreased. Mite population had increased in 8 weeks after infestation. Fluctuation of mite population depends on the condition of the host as feeds, place to take cover, and to breed. If condition of plant is fresh, mite population will increase. If quality of the host decreases, mite population will decrease. Mite population of tested clones had similar pattern. Figure 3 shows that UJ5 had the highest mite population, 228 mites/plant, while SM 1219-9 had low mite population, 160 mites/plant. Insect population has a tendency to fluctuate as effect of environment factor. Environmental factors determine the increasing or decreasing of the mite population.

3.2.2.
e Damage Intensity of the Mite Attack. In glass house, yellowish spot along leaf veins was the first symptom of mite attack. is symptom can be seen in 2 weeks after infestation. After that, symptoms spread to form necrotic zones, so that color of leaf becomes brownish yellow. On the field, mite attack was started on leaf of lower part of plant and then attack leaf of upper part of plant. If population of mite is high, the color of the attacked leaf becomes brown and dry; then the leaf falls out.
In 2 weeks after infestation, the damage intensity of the mite attack was low 8-14% with average 11.39%; there were no differences between clones in response to mite attack. In 3 weeks after infestation, the damage intensity of the mite attack increased 17-36% with average 29%. Clone OMM 0915-11 had low damage intensity, similar to UJ5d50-11 (23.40%) and CM 4867-1 (22.66%) ( Table 4).
In 4 weeks after infestation, the damage intensity reached 53%. In 5 weeks after infestation, clone UJ5d50-11 had the highest the damage intensity (69%), while clone OMM 0915-11 had the lowest damage intensity (55%), and significantly difference from the other clones. In 6 weeks after infestation, the damage intensity increased with the highest value 73%, and clone OMM 0915-11 had lowest value 59%. In 7 weeks after infestation, the damage intensity of nine clone (CId50-223, UJ5d50-11, CM 7514-7, UJ3, UJ5, UJ3d30-553-1, UJ3d30-511-2, UJ5d50-99-1, and UJ5d50-207-3) decreased because most of the leaves of those clones had fallen out. It means that those clones were more susceptible than the other clones. In 8 weeks after infestation, most clones had decreased intensity except clone OMM 0915-11, there was no fall-out leaf and dry leaf on this clone (Table 4).
In 6 weeks after infestation, there was no fall-out leaf on all clones tested, so this time was a good time to determine the response of clones to mite attack. During that time, OMM 0915-11 had low damage intensity (59%), and clone OMM0916-2, CM 4867-1, and Adira 4 had similar response to mite attack with this clone. Clone OMM0916-2, CM 4867-1, and Adira 4 were resistant, while UJ5d50-11, CM 7514-7, SM 1219-9, UJ3, UJ5, Litbang UK2, UJ3d30-553-1, UJ3d30-511-2, UJ5d50-99-1, and UJ5d50-207-3 were moderately resistant. Clone CId50-223 was susceptible (Table 5). e interest of insects in plants is a response of stimulation that comes from plants, so insects come and eat the plant. ere were five stages to find the host that was suitable for feeds, to take cover, and to breed. e finding of the host stage was affected by the light, temperature, humidity, wind, and the gravity. On finding the host stage, insects with a sense of sight and smell can find the right host, because insects were interested in stimulation from plant in the form of color, size, and shape of plant. After finding the host, stimulation of plant in the form of short distance encourages insects to stay on the plant. With the sense of touch and taste, insect will test the plant whether the plant can be accepted as the host or not. When the plant is suitable for the host, then insects will continue to eat. Table 4 shows that there were five clones with dark green leaf color and ten clones with green leaf color. ere were four clones with dense pubescence, two clones with rare pubescence, and 9 clones with no pubescence on young leaf. Clones tested had 5-7 lobes, and most clones had a lanceolate lobe. e resistance clones (OMM0916-2, CM 4867-1, and OMM 0915-11) had a lanceolate lobe, a green leaf with no pubescence, so leaves become a little rough when Note. * Varieties. touched (Table 6). is condition is not preferred by or interesting to mite as a place for eating, so mite attack on resistant clone was lower than the susceptible clone.

Discussion
e fresh tuber yield of OMM 0915-11 was the highest of 55.67 t/ha, significantly higher (58%-74%) than the existing varieties UJ3, UJ5, and Adira 4. is increasing value was equal to Rp 20.4 million to 23.6 million/ha or around US $ 1360-1576, if US $ 1 is equal to Rp 15,000 (Indonesian Rupiah). e assumption of the cost of the fresh tuber was Rp 1000/kg. On the other hand, this clone was resistant variety or highly resistant variety based on both the green house and field experiments. is clone is suitable for the area where the mite is a serious problem. e fresh tuber yields of mutant UJ5d50-99-1 and UJ5d50-207-3 were significantly higher than the original variety of them, i.e., UJ5.
at means, there is an opportunity to increase the fresh tuber yields of cassava through mutation. On the other hand, this is in line with the research result reported by Sholihin and Mejaya [7].
is promising clone needed to be tested in some environments to evaluate the adaptability of the promising   Scientifica clone [8][9][10][11].
ere was interaction between environment and genotype for the fresh tuber yield [12][13][14]. Cassava environment in Indonesia is very diverse, by which growth environment involved air temperature, kind of soil, type climate, rain fall, humidity, and so on. e starch yield is multiplication between the fresh tuber yield and starch content. e highest starch yield (10.89 t/ha) had been achieved by clone UJ5d50-207-3. e mutant clone with original variety is UJ5. So there is an opportunity to increase the starch yield through mutation breeding. is is in line with the research result reported by Sholihin and Mejaya [7]. Clone UJ5d50-207-3 was moderately resistant to mite based on the field experiment and the green house experiment. So this clone is prospectively developed in area where the mite attack was not a serious problem such as the area with wet climate, for example, Lampung. Lampung is a center of cassava production in Indonesia and most cassava are used as row material for starch factory.  Clone OMM 0915-11 was resistant to mite based on the field experiment and the green house experiment. is clone can be used as parent in cassava hybridization as source gene for high yield and mite resistance. Clone OMM 0915-11 was also in different group with release variety such as UJ3, UJ5, Litbang UK2. is means that if clone OMM 0915-11 was used as a parent in crossing block to improve the released variety, there will be high opportunity to get a good progeny. is variety can be used as source of gene for resistance to mite. e different response of clones tested to the mite attack is predicted because of presence of antibiosis mechanism that inhibits the growth and development of insect. Antibiosis can decrease the insect population, reproductive power, period of reproduction, and death of nymph. is is in line with opinion of Astuti et al. [15] that resistance of plant to insect was because of existence of antibiosis that produced toxin that could kill insect or inhibit the growth of the insect. Antibiosis can decrease the insect population by decreasing the reproductive power and time of reproduction and increasing the death of nymph. On the other hand, Iswanto et al. [16] argue that there was a tolerant genotype related to the insect resistance, which was genotype that could decrease the loss of yield. Mariscal et al. [5] reported that additive and nonadditive gene effects play a role in the expression of cassava green mite density and cassava green mite leaf damage.
is in line with what was reported by Wolfe et al. [17]. Ezenwaka et al. [18] reported that 35 single-nucleotide polymorphisms (SNPs) were significantly associated with cassava green mite severity, leaf pubescence, leaf retention, stay green, shoot tip compactness, and shoot tip size. ey were possibly an indication of pleiotropy or the presence of closely linked genes that regulate their traits.
Evaluation of cassava clone to mite attack can be done through the glass house evaluation and the field evaluation. e disadvantage of the field evaluation is difficulty in the uniformity of mite attack, while the advantage of the field  Scientifica 7 evaluation is that mite attack is similar to the real condition. According to Rahmathulla et al. [19], the mite population had a tendency to fluctuate as the effect of environment factor. e degree of influence of environment factor determines the insect population. e advantage of the green house evaluation is that uniformity of mite attack can be pursued by infestation, while disadvantage of the green house evaluation is that mite attack is not similar to the riel condition. Time for observation of the damage intensity of the mite attack is important in the selection and evaluation of cassava clone to develop a new variety for resistance to mite. Based on the green house evaluation, 6 weeks after infestation was a good time to determine the response of clones to mite attack. At that time, there was no fall-out leaf on all clones tested. Sholihin [20] reported that the time for observation of the damage intensity of the mite attack was 7 weeks after infestation. e philosophy in determining the time for observation of the damage intensity of the mite attack is the best time to distinguish the tested clone for resistance to mite. ere is another method in selection and evaluation of cassava clone for mite resistance, that is, Markers Assisted Selection (MAS). Ezenwaka et al. [4] reported that there were nine candidate genes that related to cassava green mite resistance.

Conclusion
e average of tuber yield of OMM 0915-11 was the highest of 55.67 t/ha, significantly higher than the existing variety UJ3, UJ5, and Adira 4.
is promising clone need to be tested in some different environments such as different kinds of soil, kinds of climate, and altitudes to know the adaptability of the promising clone. Clone OMM 0915-11 was resistant to mite based on both the field and the green house experiments. is clone has a chance to be released as new variety for high yield and resistance to mite. is clone also can be used as parent in cassava hybridization as source gene for high yield and mite resistance. e highest starch yield (10.89 t/ha) had been achieved by clone UJ5d50-207-3. Clone UJ5d50-207-3 was moderately resistant to mite based on both the field and the glass house experiments. So, both clones are prospective to be released as new variety for specific area such as the area with wet climate; one of them is in Lampung Province.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest regarding the publication of this study.