Effect of Prohydrojasmon on the Growth of Eggplant and Komatsuna

Article Eﬀect of Prohydrojasmon on the Growth of Eggplant and Komatsuna

Haidar Raﬁd Azis 1, Shinya Takahashi 1,2,* , Masami Koshiyama 3, Hiroshi Fujisawa 4 and Hiroko Isoda 1,2

1 Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Tsukuba 305-8572, Japan; haidar.raﬁ[email protected] (H.R.A.); [email protected] (H.I.) 2 Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan 3 Specialty Chemical Division, Zeon Corporation, Chiyoda-ku, Tokyo 100-8246, Japan; [email protected] 4 Headquarter, Zeon Corporation, Chiyoda-ku, Tokyo 100-8246, Japan; [email protected] * Correspondence: [email protected]

Received: 8 September 2020; Accepted: 13 October 2020; Published: 15 October 2020

Abstract: Prohydrojasmon (PDJ) can improve the polyphenol and anthocyanin content and antioxidant activity of some crop plants, but it also shows a suppressive effect on the plant growth. This study aimed to investigate the inhibitory effect of PDJ on the growth of two crop plants: komatsuna (Brassica rapa var. periviridis) and eggplant (Solanum melongena). We applied various concentrations of PDJ drip-wise or by spraying to eggplant and komatsuna seedlings and made detailed observations of growth. In general, no significant suppressive effect of PDJ was observed in the aerial parts in both plants. However, a significant inhibitory effect was found in roots treated with PDJ at concentrations of 600 and 1000 ppm. Interestingly, komatsuna treated with PDJ at a concentration of 200 ppm in both approaches resulted in a significant increase in root weight up to 37%. At a concentration range of 200–400 ppm, PDJ showed no inhibitory effects, and in some cases slightly promoted root growth. Therefore, this could be the recommended concentration range. We conclude that application of PDJ can still be beneficial to the vegetable crops without causing serious inhibition or suppression effects on the growth, as long as it is kept at rather low concentrations.

Keywords: prohydrojasmon; inhibitory effect; plant hormone; komatsuna (Brassica rapa var. periviridis); eggplant (Solanum melongena)

1. Introduction Jasmonates are among the most important plant hormones which play roles in many aspects of plant growth. As a class of oxylipins, jasmonates can induce a wide range of responses in higher plants. Since the first isolation of its methyl ester (jasmonic acid methyl ester), numerous jasmonates have been detected in various plant phyla. Jasmonates are found in algae, mosses, fungi, gymnosperms, and angiosperms [1–3]. Prohydrojasmon (PDJ) is a synthetic analog of jasmonic acid (JA) developed as a plant growth regulator. Plant growth regulators are defined as naturally or chemically synthesized substances that play roles in developmental or metabolic processes in plants [4]. The activity of PDJ may be similar to that of endogenous JA. It has been shown to affect several physiological processes, including senescence, leaf abscission, fruit ripening, coloration, and pigment accumulation [5,6]. Other studies have shown that PDJ functions as an elicitor in plants to stimulate the accumulation of secondary metabolites including phenolics, anthocyanins, terpenoids, and glucosinolates [7,8]. Numerous studies have reported that application of PDJ, like methyl jasmonate (MJ), can improve several aspects of crop quality. Application of PDJ has been reported to improve the hand-picking efficiency of satsuma mandarin [9]. PDJ applied to Gala and Braeburn apples a couple of days before

Plants 2020, 9, 1368; doi:10.3390/plants9101368 www.mdpi.com/journal/plants Plants 2020, 9, 1368 2 of 15 harvest resulted in a significant increase in red color index [10]. Similarly, the red-blushed color in some cultivars of mangoes significantly improved after being treated with PDJ at low concentration a few weeks prior to harvest [11]. This kind of color development was also reported to have occurred in red pear after being sprayed with PDJ during the pre-color-change period [12]. The color improvements in those fruits are strongly associated with the accumulation of anthocyanins which is enhanced by the application of PDJ. To date, we found that studies using PDJ majorly are still dominated by its application to fruit crops. In our previous study, however, we reported that application of PDJ to vegetable crops significantly increased the phenolic compounds, anthocyanin content, and antioxidant activity in komatsuna and lettuce [13]. Despite improvements of those secondary metabolites, PDJ is also known to have some kind of suppressive or inhibition effects on plant growth, particularly in initial plant growth [14]. In fact, growth inhibition was among the first characterized physiological effects of jasmonates, particularly root growth inhibition [15,16]. Previous studies have found that exogenous application of JA can inhibit various aspects of seedling growth including primary root growth, leaf expansion, and hypocotyl elongation [17–19]. One study emphasized that root growth is more sensitive to MJ [20]. Although this suppressive effect of PDJ is not as strong as that of MJ [21], it could still interrupt the plant growth, particularlyPlants if it 2020 is, applied9, x FOR PEER at REVIEW higher concentrations. To answer how far the suppressive2 of e16ff ect of PDJ could interrupt the growth of plants, here we conducted a study aimed at investigating the effect of picking efficiency of satsuma mandarin [9]. PDJ applied to Gala and Braeburn apples a couple of days PDJ at variousbefore concentrations harvest resulted onin a thesignificant growth increase and developmentin red color index of [10]. komatsuna Similarly, the and red-blushed eggplant seedlings. color in some cultivars of mangoes significantly improved after being treated with PDJ at low 2. Results concentration a few weeks prior to harvest [11]. This kind of color development was also reported to have occurred in red pear after being sprayed with PDJ during the pre-color-change period [12]. The 2.1. Effect ofcolor PDJ improvements Applied Drip-Wise in those onfruits Growth are strongly of Komatsuna associated with the accumulation of anthocyanins which is enhanced by the application of PDJ. The applicationTo date, ofwe PDJfound drip-wise that studies onusing komatsuna PDJ majorly atare thestill lowestdominated and by its highest application PDJ toconcentrations fruit (200 and 1000crops. ppm, In our respectively)previous study, showedhowever, we a tendencyreported that towards application a of slight PDJ to increase vegetable incrops shoot length significantly increased the phenolic compounds, anthocyanin content, and antioxidant activity in and its intermediatekomatsuna and concentrations lettuce [13]. (400 and 600 ppm) showed an opposite tendency towards a slight decreaseDespite in shoot improvements length. All of those results secondary regarding metabolites, this PDJ parameter is also known were to have statistically some kind insignificantof (Figure1a).suppressive The highest or inhibition concentration effects on of plant PDJ growth, drip-wise particularly (1000 inppm) initial plant showed growth a tendency[14]. In fact, towards a slight increasegrowth in inhibition leaf width, was whileamong the the first concentrations characterized physiological below that effects (200, of jasmonates, 400, and particularly 600 ppm) showed a root growth inhibition [15,16]. Previous studies have found that exogenous application of JA can tendency towardsinhibit various a slight aspects decrease of seedling in leaf growth width. including Similarly, primary all root results growth, regarding leaf expansion, this parameter and were statisticallyhypocotyl insignificant elongation (Figure [17–19].1b). One Interestingly, study emphasized PDJ drip-wise that root growth at concentrations is more sensitive of to 600 MJ and[20]. 1000 ppm significantlyAlthough reduced this root suppressive length byeffect 38% of PDJ and is 43%,not as respectively;strong as that of the MJ remaining[21], it could still concentrations interrupt the (200 ppm plant growth, particularly if it is applied at higher concentrations. To answer how far the suppressive and 400 ppm) showed statistically insignificant change in the root length (Figure1c). PDJ applied by effect of PDJ could interrupt the growth of plants, here we conducted a study aimed at investigating drip-wise approachthe effect seeminglyof PDJ at various didnot concentrations show a distinctive on the growth change and todevelopment the dimensional of komatsuna growth and of the aerial part of komatsuna,eggplant seedlings. however, at higher concentrations, the root length was significantly suppressed.

(a) (b) (c)

Figure 1. ShootFigure length 1. Shoot ( alength), leaf (a), width leaf width (b), (b and), and root root lengthlength (c () cof) komatsuna of komatsuna seedlings seedlings treated with treated PDJ with PDJ applied drip-wise at various concentrations. Komatsuna seeds were germinated and grown in soil, applied drip-wiseand PDJ was at variousapplied to concentrations.seedlings at 1 week before Komatsuna harvest. Shoot seeds length were and germinated leaf width values and are grownthe in soil, and PDJ wasaverage applied difference to seedlings between characteristics at 1 week before measured harvest. before and Shoot after lengthtreatment and (n = leaf4). Root width length values is are the average diﬀaverageerence value between (n = 4) characteristicsmeasured on the final measured day of pot before culture and(day after21). Error treatment bars indicate (n =standard4). Root length is error (SE). Asterisks (*) indicate statistically significant difference in comparison to the control using average value (n = 4) measured on the final day of pot culture (day 21). Error bars indicate standard error (SE). t-test at p < 0.05. Asterisks (*) indicate statistically significant difference in comparison to the control using t-test at p < 0.05. 2. Results

2.1. Effect of PDJ Applied Drip-Wise on Growth of Komatsuna The application of PDJ drip-wise on komatsuna at the lowest and highest PDJ concentrations (200 and 1000 ppm, respectively) showed a tendency towards a slight increase in shoot length and its intermediate concentrations (400 and 600 ppm) showed an opposite tendency towards a slight decrease in shoot length. All results regarding this parameter were statistically insignificant (Figure 1a). The highest concentration of PDJ drip-wise (1000 ppm) showed a tendency towards a slight

Plants 2020, 9, x FOR PEER REVIEW 3 of 16

increase in leaf width, while the concentrations below that (200, 400, and 600 ppm) showed a tendency towards a slight decrease in leaf width. Similarly, all results regarding this parameter were statistically insignificant (Figure 1b). Interestingly, PDJ drip-wise at concentrations of 600 and 1000 ppm significantly reduced root length by 38% and 43%, respectively; the remaining concentrations (200 ppm and 400 ppm) showed statistically insignificant change in the root length (Figure 1c). PDJ applied by drip-wise approach seemingly did not show a distinctive change to the dimensional Plantsgrowth2020 ,of9, 1368the aerial part of komatsuna, however, at higher concentrations, the root length3 ofwas 15 significantly suppressed. The tendency towards an 8% and 2% increase in the weight of the aerial parts induced by 200 The tendency towards an 8% and 2% increase in the weight of the aerial parts induced by 200 and and 1000 ppm exposure levels of PDJ, respectively, was statistically insignificant, as well as the 1000 ppm exposure levels of PDJ, respectively, was statistically insignificant, as well as the decline decline in these indices detected in the case of 400 and 600 ppm exposure levels (Figure 2a). In in these indices detected in the case of 400 and 600 ppm exposure levels (Figure2a). In contrast, contrast, PDJ applied drip-wise at concentrations of 600 and 1000 ppm reduced the root weight by PDJ applied drip-wise at concentrations of 600 and 1000 ppm reduced the root weight by 66% and 66% and 79% (both p < 0.01), respectively, at 200 ppm showed a significant increase in the root weight 79% (both p < 0.01), respectively, at 200 ppm showed a significant increase in the root weight by 26%, by 26%, and at 400 ppm showed a slight decrease tendency in the root weight by 20%, relative to its and at 400 ppm showed a slight decrease tendency in the root weight by 20%, relative to its control control (Figure 2b). When it was applied drip-wise, especially at higher concentrations, PDJ highly (Figure2b). When it was applied drip-wise, especially at higher concentrations, PDJ highly reduced reduced the weight of the root of komatsuna, and interestingly, stimulated the root growth at a the weight of the root of komatsuna, and interestingly, stimulated the root growth at a concentration of concentration of 200 ppm. However, we did not observe significant change in the weight of the aerial 200 ppm. However, we did not observe significant change in the weight of the aerial parts, but merely parts, but merely an increase or decrease tendency. an increase or decrease tendency.

(a) (b)

FigureFigure 2.2. WeightsWeights ofof aerialaerial partsparts ((aa)) andand rootsroots ((bb)) ofof komatsunakomatsuna treatedtreated withwith PDJPDJ appliedapplied drip-wisedrip-wise atat variousvarious concentrations.concentrations. KomatsunaKomatsuna seeds were germinatedgerminated and growngrown inin soil.soil. PDJ atat 200,200, 400,400, 600,600, andand 10001000 ppmppm waswas appliedapplied toto seedlingsseedlings atat 11 weekweek beforebefore harvest.harvest. WeightWeight ofof aerialaerial partsparts ((a)a) andand rootroot weightweight ((b)b) areare averageaverage valuesvalues (n(n= = 4)4) measuredmeasured onon thethe finalfinal dayday ofof potpot cultureculture (day(day 21).21). ErrorError barsbars indicateindicate standard standard error error (SE). (SE). Asterisks Asterisks (* and (* **) and indicate **) indicate statistically statistically significant significant difference indifference comparison in tocomparison the control to using the control t-test at usingp < 0.05 t-test and at pp << 0.01,0.05 and respectively. p < 0.01, respectively.

2.2. E ect of PDJ Applied by Spraying on Komatsuna Growth 2.2. Effectff of PDJ Applied by Spraying on Komatsuna Growth WhenWhen PDJPDJ waswas appliedapplied byby sprayingspraying atat concentrationsconcentrations ofof 200,200, 400,400, 600,600, andand 10001000 ppm,ppm, komatsunakomatsuna showedshowed aa tendencytendency towardstowards aa slightslight increaseincrease inin shootshoot lengthlength byby 11%, 8%, 10%, and 15%, respectively, relativerelative toto itsits control,control, whichwhich waswas statisticallystatistically insignificantinsignificant (Figure(Figure3 a).3a). In In contrast, contrast, PDJ PDJ sprayed sprayed at at 200, 200, 400,400, 600,600, andand 10001000 ppmppm showedshowed aa tendencytendency towardstowards aa slightslight decreasedecrease inin leafleaf widthwidth byby 9%,9%, 18%,18%, 4%,4%, andand 1%,1%, respectively,respectively, relativerelative toto itsits control,control, whichwhich waswas statisticallystatistically insignificantinsignificant asas wellwell (Figure(Figure3 3b).b). p SpraySpray treatmentstreatments withwith PDJPDJ atat 10001000 ppmppm highlyhighly significantlysignificantly inhibitedinhibited rootroot lengthlength byby 26%26% ((p< < 0.01),0.01), atat 600600 ppmppm showedshowed aa decreasedecrease tendencytendency inin rootroot lengthlength byby 17%17% whichwhich waswas statisticallystatistically insignificant,insignificant, atat 400400 ppm ppm showed showed an increasean increase tendency tendency in root in length root by length 8% which by 8% was which also statistically was also insignificant, statistically p andinsignificant, eventually and at 200 eventually ppm, it significantly at 200 ppm, increased it significantly root length increased by 20% (root< 0.05) length (Figure by 20%3c). Similar(p < 0.05) to the(Figure drip-wise 3c). Similar approach, to the spray drip-wise treatments approach, with PDJ sp didray nottreatments cause significant with PDJ change did not to cause the dimensional significant growthchange ofto aerialthe dimensional parts of komatsuna, growth of but aerial again parts the higherof komatsuna, concentration but again of PDJ the significantly higher concentration suppressed of the root length of komatsuna, and interestingly, we found that at a concentration of 200 ppm, it also significantly increased the root length of komatsuna.

Plants 2020, 9, x FOR PEER REVIEW 4 of 16

PDJ significantly suppressed the root length of komatsuna, and interestingly, we found that at a Plants 2020, 9, 1368 4 of 15 concentration of 200 ppm, it also significantly increased the root length of komatsuna.

Plants 2020, 9, x FOR PEER REVIEW 4 of 16

PDJ significantly suppressed the root length of komatsuna, and interestingly, we found that at a concentration of 200 ppm, it also significantly increased the root length of komatsuna.

(a) (b) (c)

FigureFigure 3. Shoot 3. Shoot length length (a), (a leaf), leaf width width ( b(b),),and and rootroot length (c (c) )of of komatsuna komatsuna seedlings seedlings sprayed sprayed with with PDJ atPDJ various at various concentrations. concentrations. Komatsuna Komatsuna seedsseeds we werere germinated germinated and and grown grown in soil, in soil, and andPDJ PDJwas was sprayedsprayed onto onto seedlings seedlings at 1at week 1 week before before harvest. harvest. Shoot length length and and leaf leaf widt widthh values values are the are average the average difference between characteristics measured before and after treatment (n = 4). Root length is average difference between characteristics measured before and after treatment (n = 4). Root length is average value (n = 4) measured on the final day of pot culture (day 21). Error bars indicate standard error (SE). value (n = 4) measured on the final day of pot culture (day 21). Error bars indicate standard error (SE). Asterisks (* and **) indicate statistically significant difference in comparison to the control using t-test Asterisks (* and **) indicate statistically significant difference in comparison to the control using t-test at p < 0.05 and p < 0.01, respectively. at p < 0.05 and p < 0.01, respectively.

The tendency towards 14%, 6%, 15%, and 13% decrease in weight of aerial parts induced by 200, 400, 600, and(a 1000) ppm exposure levels of PDJ by(b spraying,) respectively, compared to( itsc) control, was statistically insigniﬁcant (Figure4a). Spraying with PDJ at concentrations of 600 and 1000 ppm reducedFigure root weight3. Shoot by length 52% ( anda), leaf 54% width (both (bp),

Figure 4. Weight of aerial parts (a) and roots (b) of komatsuna seedlings sprayed at various concentrations. Komatsuna seeds were germinated and grown in soil. Spray treatments of PDJ at 200, 400, 600, and 1000 ppm were applied to seedlings at 1 week before harvest. The weight of aerial parts (a) and roots (b) are shown as average values (n = 4) measured on the final day of pot culture (day 21). Error bars indicate standard error (SE). Asterisks (* and **) indicate statistically significant difference in comparison to the control using t-test at p < 0.05 and p < 0.01, respectively.

The tendency towards 14%, 6%, 15%, and 13% decrease in weight of aerial parts induced by 200, 400, 600, and 1000 ppm exposure levels of PDJ by spraying, respectively, compared to its control, was statistically insignificant (Figure 4a). Spraying with PDJ at concentrations of 600 and 1000 ppm reduced root weight by 52% and 54% (both p < 0.01), respectively; at 200 ppm, significantly increased root weight by 37%; and at 400 ppm, showed a tendency towards a decrease in root weight by 13%, relative to its control (Figure 4b). Regarding the weight data, the result of spraying was identical to (a) (b) the drip-wise approach, whereas the high concentration of PDJ highly reduced the root length of Figurekomatsuna,Figure 4. 4.Weight andWeight the of oflowest aerial aerial partsconcentration parts (a )(a and) and rootsincreased roots (b )(b of)the of komatsuna rootkomatsuna length seedlings seedlingssignificantly. sprayed sprayed Similarly, at at various various no concentrations. concentrations. Komatsuna Komatsuna seedsseeds were germinated germinated an andd grown grown in in soil. soil. Spray Spray treatments treatments of PDJ of PDJat 200, at 200,400, 400,600, 600,and and1000 1000 ppm ppm were were applied applied to seedlings to seedlings at 1 atweek 1 week before before harvest. harvest. The The weight weight of aerial of aerial parts parts(a) and (a) and roots roots (b) are (b) shown are shown as average as average values values (n = 4) (n measured= 4) measured on the on final the day final of daypot ofculture pot culture (day 21). (dayError 21). bars Error indicate bars indicate standard standard error (SE). error Asterisks (SE). Asterisks (* and **) (* indicate and **) indicate statistically statistically significant significant difference diffinerence comparison in comparison to the control to the using control t-test using at t-testp < 0.05 at pand< 0.05 p < 0.01, and prespectively.< 0.01, respectively.

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Plants 2020, 9, 1368 5 of 15 significant changes were found in the weight of aerial parts of komatsuna treated by PDJ spray; every concentration only indicated a tendency towards increase or decrease. AtAt the the highest highest concentration concentration (1000 (1000 ppm), ppm), PDJ caused PDJ caused wilting wilting/shrinking/shrinking symptoms symptoms in some leavesin some ofleaves komatsuna of komatsuna on day 19 on and day day 19 and 21 of day the 21 pot of culture the pot(Figure culture5 ).(Figure These 5). symptoms These symptoms may have may been have relatedbeen torelated the onset to the of onset senescence. of senescence. Strong responsesStrong responses to PDJ into termsPDJ in of terms growth of growth and development and development were observedwere observed in the root. in the Visually, root. Visually, at lower at concentrations lower concentrations (200–400 (200–400 ppm), PDJppm), led PDJ to theled formationto the formation of a robustof a robust root network, root network, while atwhile higher at higher concentrations concentrations (600–1000 (600–1000 ppm), theppm), suppressive the suppressive eﬀect ofeffect PDJ of resultedPDJ resulted in a somehow in a somehow frail and frail fragile and fragile root network root network (Figure (Figure6). 6).

a b

Figure 5. Wilting of komatsuna leaves. Pictures were taken at day 19 (a) and day 21 (b) and show wilting of komatsuna leaves after being sprayed with 1000 ppm PDJ. Figure 5. Wilting of komatsuna leaves. Pictures were taken at day 19 (a) and day 21 (b) and show 2.3. Eﬀectwilting of PDJ of komatsuna Applied Drip-Wise leaves after on Growthbeing sprayed of Eggplant with 1000 ppm PDJ.

In eggplant, PDJ applied drip-wise at concentrations of 200, 400, 600, and 1000 ppm showed a tendency towards a slight decrease in shoot length by 14%, 3%, 1%, and 8%, respectively, relative to its control, which was statistically insignificant (Figure7a). Similarly, PDJ applied drip-wise at concentrations of 200, 400, 600, and 1000 ppm showed a tendency towards a slight decrease in leaf width by 8%, 10%, 7%, and 19%, respectively, relative to its control, which was insignificant as well (Figure7b). In terms of root length, PDJ applied drip-wise at concentrations of 200, 400, and 600 ppm showed a tendency towards a slight increase in root length by 8%, 6%, and 14%, respectively, and at 1000 ppm, showed a decrease tendency in root length by 0.3% relative to its control, which was statistically insignificant (Figure7c). Since there was no significant change observed within all of these data, it seems that the dimensional growth of eggplant was not affected by the PDJ through the drip-wise approach. All decrease or increase cases showed merely a tendency rather than stimulating or suppressing the growth by PDJ.

Plants 2020, 9, 1368 6 of 15 Plants 2020, 9, x FOR PEER REVIEW 6 of 16

c d e a b

f g h i j

Figure 6. Eﬀect of PDJ on root growth and development of komatsuna. Pictures were taken on the ﬁnal day (day 21) of pot culture of seedlings treated with 0 (a,f), 200 (b,g), 400 (c,h), 600 (d,i), and 1000 (e,j) ppm PDJ applied drip-wise (a–e) or by spraying (f–j). PDJ at concentrations of 200 and 400 ppm (b,g and c,h) resulted in more complex, thicker, and stronger root networks, and at concentrations of 600 and 1000 ppm (d,i and e,j) resulted in less complex, thinner, and weaker root networks. In general, lower concentrations (200–400 ppm) of PDJ applied drip-wise, rather than by spraying, stimulated root growth and development. PDJ applied drip-wise led to stronger and more robust roots, and better incorporation between soil and roots; PDJ applied by spraying led to weaker and more fragile roots, and a crumbly and brittle (loose) soil structure. Plants 2020, 9, x FOR PEER REVIEW 7 of 16

Figure 6. Effect of PDJ on root growth and development of komatsuna. Pictures were taken on the final day (day 21) of pot culture of seedlings treated with 0 (a and f), 200 (b and g), 400 (c and h), 600 (d and i), and 1000 (e and j) ppm PDJ applied drip-wise (a–e) or by spraying (f–j). PDJ at concentrations of 200 and 400 ppm (b,g and c,h) resulted in more complex, thicker, and stronger root networks, and at concentrations of 600 and 1000 ppm (d,i and e,j) resulted in less complex, thinner, and weaker root networks. In general, lower concentrations (200–400 ppm) of PDJ applied drip-wise, rather than by spraying, stimulated root growth and development. PDJ applied drip-wise led to stronger and more robust roots, and better incorporation between soil and roots; PDJ applied by spraying led to weaker and more fragile roots, and a crumbly and brittle (loose) soil structure.

2.3. Effect of PDJ Applied Drip-Wise on Growth of Eggplant In eggplant, PDJ applied drip-wise at concentrations of 200, 400, 600, and 1000 ppm showed a tendency towards a slight decrease in shoot length by 14%, 3%, 1%, and 8%, respectively, relative to its control, which was statistically insignificant (Figure 7a). Similarly, PDJ applied drip-wise at concentrations of 200, 400, 600, and 1000 ppm showed a tendency towards a slight decrease in leaf width by 8%, 10%, 7%, and 19%, respectively, relative to its control, which was insignificant as well (Figure 7b). In terms of root length, PDJ applied drip-wise at concentrations of 200, 400, and 600 ppm showed a tendency towards a slight increase in root length by 8%, 6%, and 14%, respectively, and at 1000 ppm, showed a decrease tendency in root length by 0.3% relative to its control, which was statistically insignificant (Figure 7c). Since there was no significant change observed within all of these data, it seems that the dimensional growth of eggplant was not affected by the PDJ through the drip-wise approach. All decrease or increase cases showed merely a tendency rather than stimulating Plants 2020, 9, 1368 7 of 15 or suppressing the growth by PDJ.

(a) (b) (c)

Figure 7.FigureShoot 7. Shoot length length (a), leaf(a), leaf width width (b (),b), and and rootroot length length (c ()c of) ofeggplant eggplant seedlings seedlings treated treated with PDJ with PDJ appliedapplied drip-wise drip-wise at various at various concentrations. concentrations.Eggplant Eggplant seeds seeds were were germinated germinated and grown and grown in soil. inPDJ soil. PDJ was applied to seedlings at 1 week before harvest. Shoot length and leaf width values are the average was applied to seedlings at 1 week before harvest. Shoot length and leaf width values are the average difference between characteristics measured before and after treatment (n = 4). Root length is the differenceaverage between value characteristics (n = 4) measured measured on the final before day of and pot after culture treatment (day 28). (Errorn = 4). bars Root indicate length standard is the average value (nerror= 4) (SE). measured on the final day of pot culture (day 28). Error bars indicate standard error (SE).

When applied drip-wise at concentrations of 200, 400, 600, and 1000 ppm, PDJ showed a tendency towards a slight decrease in the weight of the aerial parts by 7%, 13%, 3%, and 26%, respectively, relative to its control, which was statistically insigniﬁcant (Figure8a). In contrast, PDJ applied drip-wise at concentrations of 200, 400, and 600 ppm showed a tendency towards a slight increase in root weight by 26%, 6%, and 7%, respectively, and at 1000 ppm showed a decrease tendency in root weight by 28%, relativePlants to2020 its, 9, control, x FOR PEER which REVIEW was also insigniﬁcant statistically (Figure8b). 8 of 16

(a) (b)

FigureFigure 8. 8.Weight Weight ofof aerial parts parts (a) ( aand) and roots roots (b) of (b eggplant) of eggplant seedlings seedlings treated with treated PDJ applied with PDJ drip- applied drip-wisewise at atvarious various concentrations. concentrations. Eggplant Eggplant seeds were seeds germinated were germinated and grown andin soil. grown PDJ was in soil.applied PDJ was applieddrip-wise drip-wise at concentrations at concentrations of 200, of 400, 200, 600, 400, and 600, 1000 and ppm 1000 to ppm seedlings to seedlings at 1 week at 1 before week beforeharvest. harvest. Weight of aerial parts (a) and roots (b) are shown as the average value (n = 4) measured on the final Weight of aerial parts (a) and roots (b) are shown as the average value (n = 4) measured on the final day day of pot culture (day 28). Error bars indicate standard error (SE). of pot culture (day 28). Error bars indicate standard error (SE). When applied drip-wise at concentrations of 200, 400, 600, and 1000 ppm, PDJ showed a Unlike in komatsuna, PDJ applied drip-wise did not show significant change in the weight tendency towards a slight decrease in the weight of the aerial parts by 7%, 13%, 3%, and 26%, of eggplant,respectively, either relative in theto its aerial control, parts which or inwas the stat rootistically part. insignificant However, (Figure we observed 8a). In contrast, that the PDJ highest concentrationapplied drip-wise of PDJ at showed concentrations a strong of tendency 200, 400, towardsand 600 ppm weight showed reduction a tendency in the towards root, yet a slight statistically insignificant.increase in An root identical weight by pattern 26%, was6%, foundand 7%, for respectively, PDJ at 200 and ppm at showing 1000 ppm tendency showed towardsa decrease increase in particulartendency rootin root weight, weight but by then28%, again relative it wasto its not control, significant. which was also insignificant statistically (Figure 8b). 2.4. EffectUnlike of PDJ in Appliedkomatsuna, by Spraying PDJ applied on Growth drip-wise of Eggplant did not show significant change in the weight of eggplant, either in the aerial parts or in the root part. However, we observed that the highest In eggplant, PDJ sprayed at concentrations of 200, 400, 600, and 1000 ppm showed a tendency concentration of PDJ showed a strong tendency towards weight reduction in the root, yet statistically towardsinsignificant. a slight An decrease identical in pattern shoot lengthwas found by 5%,for PDJ 2%, at 3%, 200and ppm 1%, showing respectively, tendency which towards was increase statistically insignificantin particular relative root weight, to its control but then (Figure again it9 a).was Similarly, not significant. PDJapplied by spraying at concentrations of

2.4. Effect of PDJ Applied by Spraying on Growth of Eggplant In eggplant, PDJ sprayed at concentrations of 200, 400, 600, and 1000 ppm showed a tendency towards a slight decrease in shoot length by 5%, 2%, 3%, and 1%, respectively, which was statistically insignificant relative to its control (Figure 9a). Similarly, PDJ applied by spraying at concentrations of 200, 400, 600, and 1000 ppm showed a tendency towards a slight decrease in leaf width by 10%, 2%, 10%, and 1%, respectively, which was also insignificant relative to its control (Figure 9b). Application of PDJ by spraying at concentrations of 200, 400, 600, and 1000 ppm also showed a tendency towards a slight decrease in root length by 17%, 19%, 12%, and 19%, respectively, which was insignificant as well relative to its control (Figure 9c). These differences were not statistically significant but reflected a trend of PDJ applied by spraying that tends to suppress the dimensional growth of eggplant seedlings.

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200, 400, 600, and 1000 ppm showed a tendency towards a slight decrease in leaf width by 10%, 2%, 10%, and 1%, respectively, which was also insignificant relative to its control (Figure9b). Application of PDJ by spraying at concentrations of 200, 400, 600, and 1000 ppm also showed a tendency towards a slight decrease in root length by 17%, 19%, 12%, and 19%, respectively, which was insignificant as well relative to its control (Figure9c). These di fferences were not statistically significant but reflected a trendPlants of PDJ 2020 applied, 9, x FOR PEER by spraying REVIEW that tends to suppress the dimensional growth of eggplant9 seedlings. of 16

(a) (b) (c)

FigureFigure 9. Shoot 9. Shoot length length (a), (a leaf), leaf width width (b (),b), and and root root length length ((cc)) of eggplant seedlings seedlings sprayed sprayed with with PDJ PDJ at variousat various concentrations. concentrations. Eggplant Eggplant seeds seeds were were germinated germinated and and grown grown in soil,in soil, and and PDJ PDJ was was applied applied to the seedlingsto the byseedlings spraying by atspraying 1 week at before 1 week harvest. before ha Shootrvest. lengthShoot length and leaf and width leaf width values values are theare the average average difference between characteristics measured before and after treatment (n = 4). Root length is difference between characteristics measured before and after treatment (n = 4). Root length is average average value (n = 4) measured on the final day of pot culture (day 28). Error bar indicates standard value (n = 4) measured on the final day of pot culture (day 28). Error bar indicates standard error (SE). error (SE). Spray applications of PDJ to eggplant at concentrations of 200, 400, 600, and 1000 ppm showed a Spray applications of PDJ to eggplant at concentrations of 200, 400, 600, and 1000 ppm showed tendency towards a slight decrease in weight of aerial parts by 11%, 8%, 11%, and 14%, respectively, a tendency towards a slight decrease in weight of aerial parts by 11%, 8%, 11%, and 14%, respectively, whichwhich was statisticallywas statistically insignificant insignificant relative relative to to its its control control (Figure (Figure 1010a).a). PDJ PDJ sprayed sprayed at at a concentration a concentration of 1000of ppm 1000 reduced ppm reduced root weight root weight significantly significantly by 41%; by 41%; at a concentrationat a concentration of 600 of 600 ppm ppm reduced reduced root root weight highlyweight significantly highly significantly by 46%; and by at46%; a concentration and at a concentration 400 and 400 200 and ppm 200 only ppm showedonly showed a tendency a tendency towards a slighttowards decrease a slight in rootdecrease weight in root by 11% weight and by 30%, 11% respectively, and 30%, respectively, which was which statistically was statistically insignificant Plants 2020, 9, x FOR PEER REVIEW 10 of 16 relativeinsignificant to its control relative (Figure to its control10b). (Figure 10b). The result regarding weight of aerial parts in eggplant by PDJ spray was similar to that by PDJ drip-wise, in which generally they showed a decrease tendency. However, regarding root weight, PDJ spray at higher concentrations showed a significant reduction, yet the concentration of 200 ppm did not show an increase tendency like that observed in PDJ drip-wise, but rather a decrease tendency. Similar to komatsuna, no significant result was shown in dimensional growth of aerial parts in eggplant. While PDJ drip-wise did not show any significant change in all growth parameters, PDJ spray at higher concentrations reduced the root weight of eggplant significantly, similar to the komatsuna case. Regarding the root, visually, it seems that lower concentrations (200–400 ppm) of PDJ led to the formation of robust, dense, and complex root networks, while the higher concentrations (600–1000 ppm) resulted in a somehow frail and fragile root network due to the distinct suppressive effect of PDJ, which was practically identical in komatsuna (Figure 11).

(a) (b)

FigureFigure 10. Weight 10. Weight of aerial of aerial parts parts (a) (anda) and roots roots (b) ( bof) ofeggplant eggplant seedlings seedlings sprayed sprayed with with PDJ PDJ at at various concentrations.concentrations. Eggplants Eggplants were germinated were germinated and grown and grownin soil. Spray in soil. treatments Spray treatments of PDJ at of 200, PDJ 400, at 200,600, and 400, 1000 ppm600, andwere 1000 applied ppm to were the seedlings applied to at the 1 week seedlings before at harvest. 1 week Weights before harvest. of aerial Weights parts (a) of and aerial roots parts (b) are shown(a) and as roots average (b) arevalues shown (n = as 4) average measured values on the (n = final4) measured day of po ont culture the final (day day 28). of potError culture bar indicates (day 28). standardError error bar indicates(SE). Asterisks standard (* errorand **) (SE). indicate Asterisks stat (*istically and **) significant indicate statistically difference significant in comparison difference to the in controlcomparison using t-test to at the p < control 0.05 and using p < 0.01, t-test respectively. at p < 0.05 and p < 0.01, respectively.

Plants 2020, 9, 1368 9 of 15

The result regarding weight of aerial parts in eggplant by PDJ spray was similar to that by PDJ drip-wise, in which generally they showed a decrease tendency. However, regarding root weight, PDJ spray at higher concentrations showed a significant reduction, yet the concentration of 200 ppm did not show an increase tendency like that observed in PDJ drip-wise, but rather a decrease tendency. Similar to komatsuna, no significant result was shown in dimensional growth of aerial parts in eggplant. While PDJ drip-wise did not show any significant change in all growth parameters, PDJ spray at higher concentrations reduced the root weight of eggplant significantly, similar to the komatsuna case. Regarding the root, visually, it seems that lower concentrations (200–400 ppm) of PDJ led to the formation of robust, dense, and complex root networks, while the higher concentrations (600–1000 ppm) resulted in a somehow frail and fragile root network due to the distinct suppressive effect of PDJ, which was practically identical in komatsuna (Figure 11). Plants 2020, 9, x FOR PEER REVIEW 10 of 16 Plants 2020, 9, 1368 10 of 15

c d e a b

f g h i j

Figure 11. Eﬀect of PDJ on eggplant root growth and development. Pictures were taken on the ﬁnal day (day 28) of pot culture of seedlings treated with 0 (a and f), 200 (b and g), 400 (c and h), 600 (d and i), and 1000 (e and j) ppm PDJ supplied drip-wise (a–e) or by spraying (f–j). PDJ at concentrations of 200 and 400 ppm (b,g and c,h) resulted in more complex, thicker, and stronger root networks, while PDJ at concentrations of 600 and 1000 ppm (d,i and e,j) resulted in less complex, thinner, and weaker root networks. In general, lower concentrations (200–400 ppm) of PDJ applied drip-wise, rather than by spraying, stimulated root growth and development. Drip-wise application of PDJ led to stronger and more robust roots, and better incorporation between soil and roots, while spraying of PDJ led to weaker, fragile roots and a crumbly and brittle (loose) soil structure. Plants 2020, 9, 1368 11 of 15

3. Discussion We found that PDJ indeed had an effect on the growth of plants, whether it stimulated or suppressed the growth in both approaches. This effect, however, was mild, especially in terms of dimensional growth (length of shoot and width of leaves) and the weight of aerial parts. PDJ at low concentration showed a stimulatory tendency towards growth, while at high concentration showed the opposite effect of inhibition tendency. In most cases, a higher concentration resulted in significant growth suppression of the root development, particularly root weight. However, in the aerial part, we found a wilting leaves phenomenon occurred in komatsuna after they sprayed with PDJ at a high concentration (1000 ppm). This phenomenon might be associated with senescence. Senescence in plants can be defined as the degeneration process of cells, organs, or the entire organism, leading to death [22]. It has been reported that treatment with MJ can lead to plant senescence. Because MJ is volatile and odoriferous, it can be directly incorporated into the internal leaf tissues during gas exchange, and then eventually promote senescence [23]. Since PDJ is an analog of MJ [10], we expect that PDJ may have a similar effect to induce senescence. Plants that are strongly negatively affected by PDJ (especially at high concentration) might show wilting/shrinking symptoms, like in the komatsuna case of this study. However, senescence is a highly complex process controlled by multiple layers of regulation and it is an integral part of the plant lifecycle. Furthermore, senescence is not a single state, and should be understood from a temporally dynamic perspective [24]. Thus, it requires further analysis to reveal whether this wilting phenomenon is directly caused by senescence or not. Previous studies have shown that aerial plant parts, especially leaves, are affected by JA. For example, a study on Arabidopsis thaliana showed that JA application inhibited the expansion of true leaves and cotyledons. In addition, JA was shown to inhibit the activity of mitotic cyclin CycB1;2, which, in turn, repressed leaf expansion. It has been reported that the Arabidopsis COI1-JAZ-MYC2 cascade mediates the JA-induced inhibition of leaf expansion [25]. However, it seems that PDJ has less or weaker inhibition effect compared with JA, particularly as shown by the results of this study with respect to all dimensional growth parameters in komatsuna and eggplant as well. Mostly, none of these data showed a significant result, with the exception of the length of root results in komatsuna. In terms of root development, we observed that at higher concentrations (600–1000 ppm), PDJ applied drip-wise tended to show a stronger suppression effect than PDJ applied by spraying. Figure 12 shows that high concentrations of PDJ applied drip-wise resulted in less dense and a smaller amount of roots than PDJ applied by spraying. This might have happened due to the direct exposure of PDJ drip-wise, which directly targets the soil. However, interestingly, at lower concentrations (200–400 ppm), drip-wise application of PDJ tended to result in rather stronger, thicker, and more complex root networks, and caused the soil to become more compact and packed. Meanwhile, PDJ applied by spraying in general tended to result in rather fragile, thinner, and less complex root networks, and a crumbly and brittle soil structure. In addition, application of PDJ by spraying resulted in a rather crumbly and brittle soil structure, whereas the drip-wise application of PDJ tended to result in more compact and packed soil. Plants 2020, 9, x FOR PEER REVIEW 11 of 16

Figure 11. Effect of PDJ on eggplant root growth and development. Pictures were taken on the final day (day 28) of pot culture of seedlings treated with 0 (a and f), 200 (b and g), 400 (c and h), 600 (d and i), and 1000 (e and j) ppm PDJ supplied drip-wise (a–e) or by spraying (f–j). PDJ at concentrations of 200 and 400 ppm (b,g and c,h) resulted in more complex, thicker, and stronger root networks, while PDJ at concentrations of 600 and 1000 ppm (d,i and e,j) resulted in less complex, thinner, and weaker root networks. In general, lower concentrations (200–400 ppm) of PDJ applied drip-wise, rather than by spraying, stimulated root growth and development. Drip-wise application of PDJ led to stronger and more robust roots, and better incorporation between soil and roots, while spraying of PDJ led to weaker, fragile roots and a crumbly and brittle (loose) soil structure.

3. Discussion We found that PDJ indeed had an effect on the growth of plants, whether it stimulated or suppressed the growth in both approaches. This effect, however, was mild, especially in terms of dimensional growth (length of shoot and width of leaves) and the weight of aerial parts. PDJ at low concentration showed a stimulatory tendency towards growth, while at high concentration showed the opposite effect of inhibition tendency. In most cases, a higher concentration resulted in significant growth suppression of the root development, particularly root weight. However, in the aerial part, we found a wilting leaves phenomenon occurred in komatsuna after they sprayed with PDJ at a high concentration (1000 ppm). This phenomenon might be associated with senescence. Senescence in plants can be defined as the degeneration process of cells, organs, or the entire organism, leading to death [22]. It has been reported that treatment with MJ can lead to plant senescence. Because MJ is volatile and odoriferous, it can be directly incorporated into the internal leaf tissues during gas exchange, and then eventually promote senescence [23]. Since PDJ is an analog of MJ [10], we expect that PDJ may have a similar effect to induce senescence. Plants that are strongly negatively affected by PDJ (especially at high concentration) might show wilting/shrinking symptoms, like in the komatsuna case of this study. However, senescence is a highly complex process controlled by multiple layers of regulation and it is an integral part of the plant lifecycle. Furthermore, senescence is not a single state, and should be understood from a temporally dynamic perspective [24]. Thus, it requires further analysis to reveal whether this wilting phenomenon is directly caused by senescence or not. Previous studies have shown that aerial plant parts, especially leaves, are affected by JA. For example, a study on Arabidopsis thaliana showed that JA application inhibited the expansion of true leaves and cotyledons. In addition, JA was shown to inhibit the activity of mitotic cyclin CycB1;2, which, in turn, repressed leaf expansion. It has been reported that the Arabidopsis COI1-JAZ-MYC2 cascade mediates the JA-induced inhibition of leaf expansion [25]. However, it seems that PDJ has less or weaker inhibition effect compared with JA, particularly as shown by the results of this study with respect to all dimensional growth parameters in komatsuna and eggplant as well. Mostly, none Plants 2020, 9, 1368 12 of 15 of these data showed a significant result, with the exception of the length of root results in komatsuna.

a b

Figure 12. PDJ drip-wise (a) showed stronger eﬀect than PDJ spray (b ). Both pictures were taken on the ﬁnal day of pot culture (day 28). The left picture shows a smaller amount of roots and thinner roots in komatsuna treated by PDJ drip-wise at a concentration of 1000 ppm. The right picture shows relatively more remaining roots (indicated by red circle) and thicker roots in komatsuna treated by PDJ spray at a concentration of 1000 ppm.

Application of JA and its derivatives may have different effects on plants, depending on the species. In general, JA affects the formation of lateral and adventitious roots [17]. One study reported that JA upregulated the expression of ERF109, which promoted lateral root formation in Arabidopsis [26,27]. Another study found that JA enhanced adventitious root formation in petunia cuttings [28]. The changes in root structure in eggplant were similar to those in komatsuna, but the root system of eggplant was much larger than that of komatsuna. Changes in the root parts might be associated with changes in cell proliferation and cell elongation caused by PDJ [29]. Another study reported that growth inhibition is associated with the inhibition of oxygen and water uptake during germination and seedling growth caused by JA [20]. Our results show that the tendency of inhibition caused by PDJ was mild in lower conditions, and it was milder in eggplant than in komatsuna. In another perspective, it can be seen that eggplant is somehow stronger in dealing with this so-called suppressive effect of PDJ. This might be due to differences in the maturity level between these two crops. It may also be related to the high phenolics content in eggplant. Because of its phenolic constituents, eggplant is known as one of the top 10 vegetables in terms of its oxygen radical scavenging capacity [30]. A study on the distribution of total phenolic contents in eggplant reported high concentrations of polyphenols in the leaves and stems, as well as in the fruit [31]. Phenolics are found abundantly in plant tissues and are actively involved in defense against biotic and abiotic stresses [32]. Therefore, the presence of phenolics in eggplant may indicate a high level of resistance against stresses. In this study, the stress factor practically was the PDJ exposure. Eggplant, particularly that treated by PDJ spraying, did not show significant inhibitory effects in all growth parameters. Somehow it is stronger and more resistant than komatsuna when dealing with the exposure to PDJ which was expected to have an inhibition effect. However, to some extent, we still detected a trend of suppression tendency caused by PDJ, whether applied drip-wise or by spraying, yet in most cases, this effect was not significant. Among all the data of dimensional growth and weight of plant aerial parts, none of them showed significant results. Therefore, the decrease or increase that occurred can only be seen as a tendency. However, it is a good sign knowing that PDJ actually did not show a serious suppressive effect on the aerial parts of plants in general. The wilting/shrinking phenomenon that was found in komatsuna occurred only for PDJ applied as a spray while it was not found for PDJ applied drip-wise and in eggplant in any approaches used. We presumed that the inhibition effect of PDJ applied by spraying can only last for about 3–4 days and afterward this effect slowly subsides. However, at high concentrations, Plants 2020, 9, 1368 13 of 15 this inhibition effect is seemingly much stronger and eventually causes wilting leaves in komatsuna regardless of the subsided effect after four days have passed.

4. Materials and Methods

4.1. Plant Materials, Growth Conditions, and PDJ Treatments Komatsuna (Brassica rapa var. periviridis) and eggplant (Solanum melongena) seeds were purchased from the Takaii Seed Co. (Kyoto, Japan). Four seeds of each plant were sown directly into each pot (diameter = 10.5 cm and depth = 9 cm) containing soil (Metro Mix, HYPONeX Japan Co., Osaka, Japan) moistened with tap water. Five pots were used: one for each treatment, and a control treated with distilled water. The potted plants were kept in a growth chamber (LPH-214-S, NK system, Japan). The growth conditions were set to 23 ◦C and ca. 60% relative humidity with a 14 h light/10 h dark photoperiod. Light was supplied by white fluorescent lights (FHF-16EX-N-H, NEC Lighting 2 1 Ltd., Tokyo, Japan). The photosynthetic photon flux density (PPFD) was ca. 110 µmol m− s− at the top of each plantlet. Distilled water was added to all the pots evenly and regularly to maintain soil moisture. To support growth, additional fertilizer (Hyponex N-P-K = 6-5-10, HYPONeX Japan Co., Osaka, Japan), diluted 1:500, was supplied to all the pots once per week after the first true leaves had completely emerged. Jasmomeito® Ekizai (Meiji-Seika Pharma Co., Yokohama, Japan), which contains 5% of the active ingredient PDJ, was prepared at 200, 400, 600, and 1000 ppm concentrations by dilution with distilled water. These PDJ treatments were applied by two approaches. The first one was by the foliar spraying approach in which the PDJ was sprayed towards the aerial parts of the plant. The second approach was by the drip-wise approach using a pipet in which PDJ was dripped directly into the soil. The volume of PDJ applied was 1 mL in both approaches and crop plants. PDJ was applied one week prior to harvest. Distilled water served as the control. The duration of pot culture for komatsuna and eggplant was 21 days and 28 days, respectively.

4.2. Plant Growth Measurements Plant growth was carefully observed and measured on the last week of the pot culture. Shoot length and leaf width were measured one day prior to treatment and then every two days until the final day. In this experiment, shoot length refers to the length of epicotyl of the longest first true leaves, measured from the base of true leaves to the tip. Leaf width was measured at the widest part of the largest true leaf. All measurements of dimensional growth were conducted using calipers. On the final day of pot culture, all plants were collected. The roots were washed with water to remove soil. Then, the plants were cut into two parts: aerial parts and roots. The root length, root fresh weight, and fresh weight of aerial parts were measured, recorded, and further analyzed. All data presented are average values (n = 4).

4.3. Statistical Analysis All statistical analyses were conducted using Microsoft Excel. Student’s t-test was used to assess statistical differences between the assay results of PDJ treatments and controls. A value of p < 0.05 was considered significant (*) and p < 0.01 highly significant (**).

5. Conclusions In conclusion, our findings indicated that PDJ did not show a significant inhibitory effect on the growth of komatsuna and eggplant. The suppression or stimulation effect by PDJ was merely a tendency, particularly in dimensional growth as well as weight of aerial parts. Application of PDJ, therefore, can still be beneficial to improve the quality of crop plants without causing the inhibition issue to the growth of the plants. However, we noticed that special attention needs to be taken when using PDJ at rather higher concentrations, in this case, PDJ at concentrations of 600 ppm and 1000 ppm. Plants 2020, 9, 1368 14 of 15

Based on our findings, PDJ used within this concentration range may start to significantly cause adverse effects on the growth of plants, particularly on the root growth, indicated by a drastic reduction of root weight (with the exclusion of spray application to eggplant). As long as the concentration of PDJ is kept low, we expect that these kinds of inhibition effects of PDJ will not affect the growth, and probably will only show a tendency towards a slight inhibition. In contrast, there are some cases showing a tendency and even significant stimulating effect on the growth of plants when PDJ at low concentration (200 ppm) is applied. Therefore, we suggest that it is much safer and better to use PDJ at low concentrations and based on this study, we propose PDJ in the concentration range of 200–400 ppm as the recommended concentration.

Author Contributions: H.R.A. and S.T. carried out the experiments. M.K., H.F., and H.I. contributed to the analysis and discussion. H.R.A., S.T., and H.I. prepared the manuscript. S.T., M.K., H.F., and H.I designed the project. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by funds from the Nihon Zeon Corporation, Tokyo, Japan. Acknowledgments: The authors are grateful to the Isoda-lab members for their useful comments. The authors thank Jennifer Smith, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac) for editing the English text of a draft of this manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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