Article Interaction between Trichoderma sp., Pseudomonas putida, and Two Organic Amendments on the Yield and Quality of Strawberries (Fragaria x annanasa cv. San Andreas) in the Huaral Region, Peru Lucero Huasasquiche 1 , Thania Ccori 2, Leonela Alejandro 2, Héctor Cántaro-Segura 3 , Tomás Samaniego 1 and Richard Solórzano 4,5,* 1 Estación Experimental Donoso, Dirección de Supervisión y Monitoreo de las Estaciones Experimentales, Instituto Nacional de Innovación Agraria (INIA), Lima 15200, Peru; lucero.26.lhs@gmail.com (L.H.); td.samaniego@gmail.com (T.S.) 2 Facultad de Agronomía, Universidad Nacional Agraria La Molina (UNALM), Lima 15024, Peru; thaniaccori@gmail.com (T.C.); leonelak2@gmail.com (L.A.) 3 Departamento de Fitotecnia, Facultad de Agronomía, Universidad Nacional Agraria La Molina (UNALM), Lima 15024, Peru; hcantaro@lamolina.edu.pe 4 Centro Experimental La Molina, Dirección de Supervisión y Monitoreo de las Estaciones Experimentales Agrarias, Instituto Nacional de Innovación Agraria (INIA), Lima 15024, Peru 5 Facultad de Ciencias Ambientales, Universidad Científica del Sur (UCSUR), Lima 15067, Peru * Correspondence: investigacion_labsaf@inia.gob.pe Abstract: Strawberry cultivation holds significant economic and social promise within Peruvian fruit production. However, conventional management practices have led to the excessive use of agrochemicals in this crop. This study proposes an organic approach to strawberry production, integrating less environmentally harmful technologies. The aim was to assess microbial inoculation by using Trichoderma sp. and Pseudomonas putida and the application of organic amendments on Citation: Huasasquiche, L.; Ccori, T.; strawberry seedlings of the commercial cultivar “San Andreas”. A field experiment was established Alejandro, L.; Cántaro-Segura, H.; with evaluations in the vegetative and productive stages. Results indicate that the co-inoculation of Samaniego, T.; Solórzano, R. Trichoderma sp. and Pseudomonas putida increased leaf area by 7%, and enhanced the aerial part’s fresh Interaction between Trichoderma sp., and dry biomass by 13% and 28%, respectively, compared to treatment without microbial inoculation. Pseudomonas putida, and Two Organic Concurrently, compost application increased the leaf number and aerial dry biomass by 22% and 19% Amendments on the Yield and Quality at the end of the vegetative stage, respectively, compared to treatment without organic amendment. of Strawberries (Fragaria x annanasa cv. San Andreas) in the Huaral Region, In addition, it reduced the days for flowering, maintaining the fruit’s physicochemical attributes. Peru. Appl. Microbiol. 2024, 4, Regarding yield, the amendments application significantly enhanced fruit weight per plant by 40%, 1110–1123. https://doi.org/10.3390/ especially when applied together with Trichoderma sp., and co-inoculation increased the number applmicrobiol4030075 of fruits per meter square by 22%. These findings highlight the potential of technologies such as microbial inoculation and organic amendments to enhance strawberry yields and to gradually reduce Academic Editor: Ian Connerton the use of synthetic fertilizers. Received: 20 May 2024 Revised: 20 June 2024 Keywords: Fragaria sp.; microbial inoculants; compost; leaf litter; Trichoderma sp.; Pseudomonas putida Accepted: 27 June 2024 Published: 22 July 2024 1. Introduction Copyright: © 2024 by the authors. Strawberry (Fragaria sp.) is one of the most appreciated cultivated berries in the world Licensee MDPI, Basel, Switzerland. due to its high economic and nutritional value [1]. In 2020, global strawberry production This article is an open access article value reached USD 14 billion [2]. Moreover, strawberry consumption provides essential distributed under the terms and nutrients and high vitamin C and folate levels. In addition, strawberries are rich in bioactive conditions of the Creative Commons compounds such as phenolic compounds, which together with vitamin C act as antioxidants Attribution (CC BY) license (https:// in the human diet [3]. creativecommons.org/licenses/by/ In 2022, more than 9.5 million tons were produced globally, with China and the United 4.0/). States leading production [2]. Latin America contributed 5% of the total production, with Appl. Microbiol. 2024, 4, 1110–1123. https://doi.org/10.3390/applmicrobiol4030075 https://www.mdpi.com/journal/applmicrobiol Appl. Microbiol. 2024, 4 1111 Peru being the third largest producer with almost 46,000 tons of volume [2]. Peruvian production value doubled during 2020–2022. Sowed areas are concentrated in the coastal regions, mainly in Lima and La Libertad, but in recent years cultivation has expanded to the Andes regions or inter-Andean valleys [4]. In addition, strawberry cultivation generates a considerable labor demand for harvesting, thus providing work opportunities throughout the year. Most strawberry fields in Peru are managed by small producers, typically cultivating areas of no more than three hectares [4], and they often receive minimal technical and financial assistance [5]. The main source of information that strawberry growers have regarding chemical input use is through sales personnel in commercial houses [6], which in many cases causes the excessive use of agrochemicals. This global problem has negatively impacted the environment, leading to ecosystem degradation. In 2018, total global emis- sions from agriculture and related land use reached 9.3 billion tons of equivalent CO2 [7], which contributes the most to the greenhouse effect. N2O emissions are expected to increase between 30–60% until 2030 [8] and fertilizer consumption by developing countries will exceed 75% of world consumption in 2050 [9]. Furthermore, the excessive use of fertilizers has led to a significant reduction in the economic efficiency of strawberry production [10]. In this context, bio-input use emerges as an alternative to generate cleaner and more sustainable production systems [11]. Among practices associated with bio-input use, the incorporation of plant-growth-promoting microorganisms into the soil can provide nutrients to plants, stimulate their growth, or protect them from pathogen attack [12]. Thus, microbial inoculation reduces the need for fertilizers, an increasingly important strategy being adopted worldwide. Trichoderma is a genus widely known for its antagonistic capacity; however, some strains of Trichoderma spp. have beneficial effects on plant growth [13]. These strains operate through various mechanisms, including the biological control of phytopathogens, improvement in nutrient absorption, increased root hair formation, and the induction of systemic resistance in plants [14]. Likewise, Pseudomonas species influence plant growth due to their ability to produce siderophores, solubilizing phosphorus and secreting antagonistic compounds for plant pathogens [15]. Solid waste management through organic composting is also a potential alternative for agricultural system sustainability. Organic amendments are used to improve the physical, microbiological, and chemical conditions of soils thereby increasing plant nutrient availability [16]. Thus, compost is an excellent organic fertilizer because it provides essential nutrients and organic matter. Additionally, compost enhances the soil’s water retention capacity, improves tillage, promotes better aeration for seed germination, and consequently supports robust plant root development [17]. Undoubtedly, the strawberry is a crop with great export potential and profitability in Peru [18]. Still, there is a need to explore new technologies that are environmentally friendly to our soil integrity and human health. Therefore, the objective of this study is to evaluate the influence of microbial inoculation and the application of organic amendments on strawberry (Fragaria x ananassa cv. San Andreas) yield and quality, as organic alternatives for a more sustainable management of this crop. 2. Materials and Methods 2.1. Plant Material and Sowing Strawberry plants (Fragaria sp. var. San Andreas) were obtained from the National Vegetable Research Program (PNIH) of the National Institute for Agrarian Innovation (INIA), as 3-month-old bare-root seedlings. Field installation was carried out in June 2023 at the Donoso Agricultural Experimental Station of INIA in Huaral Valley, and the experiment lasted seven months, from transplanting to harvest. The Huaral region is influenced by winter fogs and drizzles, creating a cool and humid environment in the middle of the desert. During the period from June to December 2023, the average temperature was 19.5 ◦C (max. 23.3 ◦C, min. 17.0 ◦C) and the relative humidity was 80.5%. The experimental area was 172.8 m2. The soil of the experimental plot was sandy clay–loam in texture, low in organic Appl. Microbiol. 2024, 4 1112 matter (0.7%), with 8.0 soil pH; the available N, P, and K were 15, 109, and 150 kg ha−1, respectively. The planting frame during transplanting was 0.8 m between furrows and 0.2 m between plants, with a total density of 62,500 plants per hectare. The field was irrigated 1–2 times per week, according to demand, and manual weeding was carried out every two weeks. No synthetic fertilizers or agrochemicals (for pest control) were used during the experimental area management. 2.2. Design and Treatments A Completely Randomized Block Design (CRBD) experimental plot was set up with a 4 × 3 factorial scheme, involving four combinations of microorganisms and three organic amendments. A total of 12 treatments were used and considering that 3 blocks were installed in the plot, a total of 36 experimental units were managed during the whole research. The different treatments used in the experiment are described in Table 1. Table 1. Evaluated factors and treatments. Factor 2. Organic Treat. Factor 1. Microbial Inoculation Amendments Application T1 Without amendments (WA) T2 Without microbial inoculation (WM) Compost (C) T3 Manure + Leaf litter (M) T4 Without amendments (WA) T5 Trichoderma viride (T) Compost (C) T6 Manure + Leaf litter (M) T7 Without amendments (WA) T8 Pseudomonas putida (P) Compost (C) T9 Manure + Leaf litter (M) T10 Without amendments (WA) T11 Trichoderma viride + Pseudomonas putida (T + P) Compost (C) T12 Manure + Leaf litter (M) The organic amendments application (compost and cow manure + avocado stubble) was carried out five days before sowing, at a 15 t ha−1 for compost, 7.5 t ha−1 for manure, and 7.5 t ha−1 for avocado stubble dose. The pH of the organic amendments was around 8.0 and the electrical conductivity was low for the manure and compost (<1 dS·m−1) but high for the leaf litter (2.2 dS·m−1). The C:N ratios for the compost, manure, and leaf litter were 10.0, 19.8, and 26.8, respectively. Microbial inoculation was carried out 4 times during the trial; the first at sowing and the remaining as complementary inoculations in the field at 21, 50, and 77 days after transplanting (dat). The first inoculation consisted of immersing the roots inside the inoculant for 15 min, and then transplanting the seedlings in the field [19], ensuring that the crown was at ground level. Complementary inoculations were carried out by adding 2 L of the inoculant to each plant’s neck [20]. 2.3. Inoculant Preparation For inoculation with Trichoderma sp., a spore suspension obtained by washing sporu- lated broken maize with this fungus (Soluciones Agrosostenibles S.A.C., Trujillo, Peru, 1 × 1012 conidia per kg concentration,) in non-chlorinated water at 40 g·L−1 was prepared (Solution A, inoculant). For inoculation with Pseudomonas putida, a pure culture of P. putida strain PS168 (private collection) was prepared, from which a 1% (v/v) dilution was made for the inoculation. Pure culture was prepared by incubating the strain in nutrient broth for 4 days at 28 ◦C until a 109 CFU mL−1 concentration (Solution B) was obtained. Sub- sequently, Solution B was diluted by taking a 10 mL aliquot and mixing it with 1 L of Appl. Microbiol. 2024, 4 1113 non-chlorinated water (Solution C, inoculant). For treatments containing both microorgan- isms, the inoculant was prepared at a rate of 10 mL of pure culture of Pseudomonas putida (Solution B) for each 1 L of spore suspension (Solution A). 2.4. Biometric Parameters The seedling mortality percentage was assessed at 30 dat. In addition, on five randomly marked plants per treatment, the leaf number, plant height (cm), and leaf area (cm2) were assessed monthly until 90 dat. The leaf number was assessed by manually counting the leaves on each plant; the height was measured from the plant’s crown to the last point of plant biomass, without spreading the leaves; and the leaf area was calculated by measuring the width and length of each leaf on the plant. Three plants were randomly removed to assess the fresh and dry biomass at 90 and 160 dat. For this, the root part was separated from the aerial part and the fresh weight of each one was recorded by a high precision balance (Axis Aka 4200, Gdañsk, Poland). They were then placed in paper bags and dried initially at room temperature for three weeks, followed by further drying in a drying oven (Yamato Scientific DS-64, Santa Clara, CA, USA) at 70 ◦C for 3 days [21]. 2.5. Performance and Quality Parameters From 90 dat onwards, plants were evaluated daily to observe the appearance of the first flower in each treatment, thereby calculating the number of days to flowering. In addition, in five randomly marked plants in each experimental unit, the number of flowers, the fresh weight (g), and the fruit dimensions (cm) were evaluated weekly. Additionally, the number of commercially ripe fruits (minimum 75% of the total fruit surface with red coloring) per experimental unit was recorded. At 160 dat, the fruits were harvested. The cell juice pH was evaluated using a multi-parameter meter (Hanna HI2020, Hanna Instruments, Leighton Buzzard, Bedfordshire, UK) by the AOAC 981.12 method; the acidity by titration with 0.1 N sodium hydroxide (AOAC 942.15 method); the Brix degrees by using a digital refractometer (PAL-1 Atago 3810, ATAGO, Tokyo, Japan); and the firmness by using a penetrometer (Lutron FR-5120, Lutron, New York City, NY, USA). 2.6. Statistical Analysis The results were analyzed using R software version 4.3.1 (Lucent Technologies, Murray Hill, NJ, USA), employing the analysis of variance (ANOVA) with a significance level of 0.05, following verification of data normality assumptions and homogeneity of variances. Means were analyzed by the least significant difference test (LSD Fisher) with a p ≤ 0.05. 2.7. Principal Component Analysis A principal component analysis (PCA) was conducted using PAST 3.24 software to assess the relationship between the studied parameters. Separate PCA analyses were performed for each factor: microbial inoculation and application of organic amendments. 3. Results 3.1. Strawberry Plant Rooting Percentage (Fragaria sp. var. San Andreas) The results indicated no interaction between the study factors; however, a significant effect of microbial inoculation on the percentage of strawberry seedling mortality was ob- served. Plants inoculated with Trichoderma sp. and co-inoculated with Trichoderma sp. and Pseudomonas putida showed lower plant mortality (p-value 0.024) compared to non-inoculated plants (Figure 1). Appl. Microbiol. 2024, 4, FOR PEER REVIEW 5 Appl. Microbiol. 2024, 4, FOR PEER REVIEW 5 Appl. Microbiol. 2024, 4 1114 Figure 1. Mortality rates in strawberry seedlings c v. San Andreas at 30 dat according to the microbial Finigoucurela1t.ioMno (rWtaMlit y= rwatiethsoinuts tmraiwcrboebrirayl isneoecdulilnagtisocnv, .TS a=n inAoncudlraetaisonat w30ithd aTtraichcodredrimnga stop.t,h Pe =m iincorocbuilal- Fitniigouncur wela 1itt.ih oM nPo(srWetuaMdliotmy= orwanitateshs o piunt simdtraai,c wrTob b+ei raPrl yi=n s oceoceu-dillnainoticgousn lc,avTti. o=Snai)nn.o AMcuneldanrteisoa nws waitthi 3t h0d Tdifrafiect hraeocndcetor rlmedtaitnsegprs .t, oaP rt=he eisn tmaoticicusrtloiacbtaiiolalnly indoifcfuerlaentito (nL (SWDM Fi s=h weri,t hαo =u t0 .m05ic.)r obial inoculation, T = inoculation with Trichoderma sp., P = inocula- twiointh wPistehu dPosmeuodnoams pountaisd ap,uTti+daP, T= c+o P-i n=o ccou-liantioocnu)l.aMtioenan).s Mweitahnds iwffeitrhe ndtilfefettreernsta lreettsetarsti satrieca slltyatdisitfifcearellnyt d(LifSfeD Fisher, α = 0.05.)3.2. rNenutm (LbSeDr o Ff iLsheaevr,e αs, =P 0la.0n5t. )H eight, and Leaf Area 3.2. NAu msibgenrifoifcLaenatv ienst,ePralacntitoHne oigf hfta,catnodrsL weaafsA orea3.2. Number of Leaves, Plant Height, and Leaf Arbeas erved in the initial evaluations for leaf area (p-vaAluesi 0g.n0i4fi8c)a anntdi nptlearnatc thieoing of factors was observed in the initialA significant interaction ohf fta (cpt-ovrasl uwea 0s .o0b27se) r(vFeigdu irne t 2h)e. iTnhitei aclo emvabli enveadl utraetaiotmnsenfot rofle ianf- aorceuala(pti-ovna lwueith0 .T04ri8c)haodnedrmpala snpt. hpeluigsh cto(mp-pvoaslut ein0c.027) (Figure 2). The c uoamtiboinnse fdort rleeaatfm areenat (opf-vinaoluceu l0a.0ti4o8n) wanidth pTlraincht ohdeeirgmhat (spp-.vpalluues 0co.0m27p)o (F origpuorrea t2io).n T whea sc othmeb minoesdt strigeantificant inter- action for both variables. It was also observed thastt,i innc boorptho rpaatriaomn ewtearsst, htheem oorgsat msiegnt of in- oculation with Trichoderma sp. plus com nic naimficeanndt- imnteenrtasc taipopnlifcoartboth variables. It was als poosotb isnecrovrepdorthataito,nin wbaost thhpe amraomst estiegrnsi,ftihcaenot rignatenri-c aacmtioennd fomr ebnottsha v ioarni ahbalse sa. Ivte wrya sv aalrsioab olbes eefrfveectd w thhaetn, i nin bteortahc ptianrga mweitthe rTs,r itchheo oderrgmana iscp a.m Fiegnudr-e 2 shows that Trpipchliocdaetrimona hspa.s ian vceormybvianriable effect whements application has a very variable efafeticot nw wheitnh icnotwer am nainnuterrea pctliunsg awvoitchadTori cshtuodbebrlme apsrpe-. Fseignutsre v2ersyh olowws tvhaaltuTersi;c hhoodweremvaers,p w. ihnecno cmobminbaintieodn wwiitth c coting with Trichoderma sp. h cowmpmoasnt,u trheep hluesigahvto acnaddo les Ftiugbubrlee 2p rsehsoewntss tvhearty Tlroiwchovdaerma s af area values increase. Thisl useigs;nh po. wine vceorm, wbihneanticoonm wbiitnhe dcowi tmanure plus avocado stubble pre- sents very low values; howifiecvaenrt, iwntheerna cctoiomnb winaesd n woti tmh a hinctoaminpeods tu,nthtiel thheeig ehntda nodf tlheea ftrairaela. values increase. This significant interaction was not macinotmaipnoesdt,u tnhteil htheiegehnt danodf tlheeaft rairael.a values increase. This significant interaction was not maintained until the end of the trial. Figure 2. Interaction between microbial inoculation and the organic amendments application on Figure 2. Interaction between microbial inoculation and the organic amendments application on the t(hae) l(eaa)f laeraefaa arte a30a tda3t0 adnadt (abn)d pl(abn)tp hlaeingthht eaitg 6h0t datat6 o0fd satrtaowfbs etrraryw pblearnryts pcvla. nStasnc Av.nSdarneaAs natd. r(eWasMa t=. F(wWigiutMhroe=u 2tw. mIintihtceorrouabtctimaioli nicn rbooebctuiwalaleteiinoo nmc, uiTcl ra=ot ibioninao,lc iuTnloa=ctiuionlnao tcwiuoinltah at inTodrni ctwhhoeidt oherrmgTraain csihpco .ad, mePr me=n aidnsmopc.eu,nlPatst= iaopinnp wolicciutahltai Potinsoe noundwo tmhiteoh- (Pnas)a eslue pdauof tmaidroaen,a aT sa tp+ u 3P0ti =dd aac,otT -ai+nndoPc (u=bl)ac poti-loainnn,ot W chueAliag t=hio twn ai,ttW h6o0Au dt= aotwr ogifat hsntoircua atwmobreegnrardnymi cpelaanmntste,s n Ccdv m=. wSeanintths A, cCnodm=rpewaoisst hta, tcM. o( mW= pwMoi st=th , wMmita=hnowuuritet hm amnicdra onlebuairafe ll iaitnnteodrc.ul)e laaftiloitnte, rT.) = inoculation with Trichoderma sp., P = inoculation with Pseudomo- nas putida, T + P = co-inoculation, WA = without organic amendments, C = with compost, M = with manuRrReee aggnaadrr ddleiiannfgg li ttththeeer. )ii nnddiivviidduuaall eeffffeeccttss ooff ttrreeaattmmeenntt oonn eeaacchh ppaarraammeetteerr,, iitt wwaass oobbsseerrvveedd iinn TTaabbllee2 2 tthhaatt,, ffoorrleaf number, microbial inoculation did not differ from the non-inoculated treatRmeegnatr;diinngfa t hleeaf nct, tihned umber, tirveidatumal m eifcferoctbsial inocuent with oTf rtircehaotdm laetiermn on did at ospn .eha ncoah t dp dif thaer faemr from the non-inoculated loweteesrt, ivta wluaes aotb9s0erdvaetd. iInn Ttarebaletm 2 etnhta;t i,n fo fra clet,a tfh neu tmrebatemr, emnitc wroibthia Tl rinicohcoudelarmtioa ns p. had thethis period, it was observed that the compost applidciadti noonti dni flfoewr fersot mva tlhuee naot n90-i ndoact. In this creased the number ofulelaatveeds treatment; in fact, the treatment with Trichoderma sp. had the lowest value at 90 dat. In this Appl. Microbiol. 2024, 4, FOR PEER REVIEW 6 Appl. Microbiol. 2024, 4 1115 period, it was observed that the compost application increased the number of leaves (p- value 0.047), differing from plants without the organic amendment. None of the factors (hpa-vda alu seig0n.0if4i7c)a,ndt iefffefericnt gonfr opmlanptl ahnetisghwti atht o9u0t dtahte. organic amendment. None of the factors had a significant effect on plant height at 90 dat. Table 2. Number of leaves, plant height, and leaf area of strawberry seedlings cv. San Andreas at Tthabe leen2d. oNf tuhme bvergeotfatlievaev estsa,gpel a(9n0t dheaitg).h (tW, aMn d= lweaitfhaorueta moficsrtorbaiwalb ienrorcyuslaeetidolnin, Tgs =c ivn.oScuanlatAionnd wreiaths aTtrtichheodeenrdmoa fstph.,e Pv e=g ientoactuivleatsiotang we i(t9h0 Pdsaetu)d. o(mWoMnas= pwutiitdhao, uTt +m Pic =ro cboi-ailnioncoucluatliaotnio, nW, TA == iwnoitchuoluatti oorn- wgaitnhicT raimcheonddermmeanstps.,, CP == winiothc ucloamtiopnoswt,i tMh P= sweuidtho mmoannaus rpeu taindad, lTea+f Plit=tecro.)- inoculation, WA = without organic amendments, C =Nwuimthbceorm opfo Lste,aMve=sw ith maPnluarneta Hndeliegahftl itter). Leaf Area Treatment Nupmerb ePrlaonf tL eaves Pla(nctmH)e ight Le(acfmA2r)e a InTtreeraatcmtieonnt pne.rsP. lant n(c.sm. ) (ncm.s2. ) Interaction Fanct.so.r 1. Microbial inocunla.st.ion n.s. WM 13.06F ±a c1to.6 a abr 1 . Microbial1in1.o8c9u l±a t1io.2n n.s. 788 ± 128 WT M 101.43.40 6± ±2.31. 6b a 1110.8.793± ±1 1.2.3n .s. 768380± ± 112480a cb PT 131.02.4 4± ±2.2. 3a b 110.2.763 ±± 11.4.3 668360 ± 113430 bcc T +P P 131.43.52 4± ±3.42. 2a a 1121.1.276 ±± 11.2.4 688467± ± 13634 bac T + P Fact1o3r. 425. ±Or3g.4 a anic amendme1n2.t1 7ap±p1li.c2ation 847 ± 164 a WA F1a1c.5to3r ±2 .1O.4r gb anic amend1m1e.1n6t a±p 1p.l2ic nat.sio. n 732 ± 158 n.s. WA 11 bC 14.1.25 3± ±2.71. 4a 11.16 ± 1.2 n.s. 732 ± 158 n.s. C 14.12 ± 2.7 a 11.82 ± 1.4 780 ± 161 11.82 ± 1.4 780 ± 161 MM 121.20.00 0± ±3.30 ab .0 a b 1111.5.555 ±± 11.4.4 770011 ±± 116677 MMeeaannssw withitdhi fdfeirfefnertelenttte lrestitnerths einsa tmhee fsaactmorei nfathcteosra mine tchoelu smanmaer ecsotlautimstinca allryed siftfaetriesntitc(aLlSlDy dFiisfhfeerr,ent= (0Lα .0S5D); nF.iss.:hneor,s αig n=i fi0c.0a5n)c;e n. .s.: no significance. TThhee ccoo--iinnooccuullaatteedd ttrreeaattmeenntt ((TTrriicchhooddeerrmmaa sspp.. ++ PPsseeuuddoommoonnaass ppuuttiiddaa)) sshhooweedd ppllaannttss wiitthh ggrreeaatteerrl eleaaffa areraeac ocmompparaerdedto toth tehoet ohtehretrr etaretmatemnetsn(tFs i(gFuigreu3re); 3th);i sthriess ureltsuwlat swsaigsn siifigcnainfit- actan60t a(pt -6v0a l(up-ev0a.l0u0e5 0) .a0n0d5)9 a0ndda 9t0(p d-vata l(up-ev0a.l0u1e2 )0..012). FFiigguurree 33.. LLeeaaff aarreeaa aaccccoorrddiningg toto mmicicrorobbiaial lininoocuculaltaitoionn frformom 303 0tot o909 0dadta. t(.W(MW M= w=itwhoituhto muticmroicbrioa-l biniaolcuinlaotciuolna,t iTo n= ,inTo=cuilnaoticounl awtiiothn TwriicthhoTdreircmhoa dseprm., aP s=p i.n, oPcu=laitniooncu wlaittiho nPsweuitdhomPosneuasd opmutoindaas, Tp u+t iPd a=, co-inoculation.). Means with different letters are statistically different (LSD Fisher, α = 0.05.); n.s. no Tsi+gnPif=icacon-cien.o culation.). Means with different letters are statistically different (LSD Fisher, α = 0.05.); n.s. no significance. 33..33.. Frreesshh aandd Drryy Biioomaassss A ssiigniifificcantt iintterraccttiion off ffaccttorrss wass obsserrved iin tthe aerriiall ((p--vallue 0..040)) and rroott ((pp--vvaalluuee0 0.0.01144))d dryryb iboimomasassps aprarmaemtertse.rsT.h Tehtere tartematemntewnth wosheosseee dseliendglsinwgesr ewceor-ei ncoc-uinlaotceud- and had compost application stood out in both parameters (Table S1). This interaction was only significant in the first evaluation (90 dat) and was not maintained towards the end of the trial, so the results were analyzed according to each study factor. Appl. Microbiol. 2024, 4, FOR PEER REVIEW 7 lated and had compost application stood out in both parameters (Table S1). This interac- tion was only significant in the first evaluation (90 dat) and was not maintained towards Appl. Microbiol. 2024, 4 the end of the trial, so the results were analyzed according to each study factor. 1116 It was observed that microbial inoculation had statistical differences in the fresh (p- value 0.029) and dry biomass (p-value 0.018) of the aerial part at 90 dat (Table 3). In both parameIttewrsa, scoob-isneorcvueldattehda tsemediclrinobgisa l(Tinriocchuodlaertimoan shpa.d +s tPasteisutdicoamlodniafsf epreunticdeas) ipnrethseenftreeds h hi(gph-vera laueeri0a.l0 2b9io)manadss,d aryndb iionm thases s(ppe-vciafilcu eca0s.e0 1o8f) aoefrtiahle dareyr ibaliopmaratssa,t t9h0e dcaot-i(nToacbulleat3i)o.nI n trebaottmh epnatr awmaes tsetrast,icsoti-cianlolycu dlaiftfeedresnete dfrloinmg sth(Ter ticrehaodtmeremnat swp.it+hoPuste uindoomcuolnaatsiopnu.t iTdha)e porregsaennitce d amhiegnhdemr eanetrsia alpbpiloimcaatisosn, ,a snpdeciinficthaellys pcoecmifipcoscta, sweaosf aabelrei atol dinrcyrebaisoem thases a,etrhiealc dor-yin boicoumlaatsiso n (pt-rveaaltume e0n.0t0w8)a sats t9a0ti sdtaict a(lTlyabdlief f3e)r,e cnotmfrpomaretdh etotr ecoatwm menatnwuirteh aonudt itnhoec utrleaatitomne.nTth we iothrgoaunt ic amamenednmdmenetn (tTsaabplpe l3ic)a. tion, specifically compost, was able to increase the aerial dry biomass (p-value 0.008) at 90 dat (Table 3), compared to cow manure and the treatment without Taabmlee n3.d Fmreesnht a(nTda bdlrey3 b).iomass of the aerial part based on microbial inoculation and the organic amendments application. (WM = without microbial inoculation, T = inoculation with Trichoderma spT.,a Pb l=e i3n.oFcurelasthioann dwidthry Pbseioumdoamsosnoafs tphuetiadear, iTal +p Pa r=t cboa-siendocounlamtioicnr,o WbiaAl i=n wociuthlaotuiot noragnadnitch aemoergnadn- ic maemntesn, Cdm = ewnittsha cpopmlicpaotsiot,n M. ( W= wMit=h wmiathnouurte manicdr olebaiaf lliitntoerc.u) lation, T = inoculation with Trichoderma sp., P = inoculation with Pseudomonas putida, T + P = co-inoculation, WA = without organic amendments, C = wi Fresh Biomass Dry Biomass Treattmh ceonmt p ost, M = with manure and leaf litter). 90 dat Harvest 90 dat Harvest Treatment Factor 1. MFircersohbBiailo imnaoscsulation Dry Biomass WM 37.8 ± 990.7d aabt 90.3 ±H 1a3r.v2e nst.s. 7.6 9±0 2d.3a tb 25.2 ±H 5a.r8v ens.ts. T 34.3F a±c 6to.0r 1b. Microbiaab 9 l2in.2o c±u 2la6t.i5o n 8.3 ± 1.4 b 25.8 ± 5.3 PW M 33.337 ±.8 6±.29 b.7 8950..33 ±± 2163..26 n.s. 8.71. 6±± 1.25. 3 b b 25.2 ± 5. T 34.3 ± 6.0 b 92.2 ± 26.5 8.3 ± 1.4 b 232.95 .±8 ±5.2 8 n.s.5.3 T + PP 42.733 ±.3 8±.5 a6. b 109.4 ±± 26.4 9.7 ±± 1.5 a2 85.3 26.6 8.1 1.5 b 2923 ±.9 6±.1 5.2 T + P Factor 2. 4O2.r7g±an8i.c5 aamendm1e0n9t.4 a±pp2l6i.c4ation 9.7 ± 1.5 a 29 ± 6.1 WA F3a4c.t5o r± 25..O8 rng.asn. ic am9en5d.2m ±e n16t .a9p npl.isc.a tion 7.9 ± 1.4 b 25.0 ± 4.4 n.s CW A 403.47.5 ±± 8.59.8 n.s. 935.24 ± 2146.91 n.s. 9.75. 9±± 1.17. 4a b 2275..20 ± 64..24 n.s M C 35.54 0±.7 8±.9 8.9 939.3 .4± ±342.64 .1 7.96. 5±± 2.10. 7b a 252.87 .±2 ±6.66 .2 b Means withM different letter3s5 i.5n ±the8 .9same facto9r3 i.n3 ±the3 4s.a6me colum7n.6 a±re 2s.t0atistically di2f5fe.8re±nt6 (.L6SD FisMheearn, sαw =i t0h.0d5if)f;e rne.nst.:l entote rssiginnitfhiecasanmcee. factor in the same column are statistically different (LSD Fisher, α = 0.05); n.s.: no significance. 3.4. Flowering, Fruit, and Yield Analysis 3.4. Flowering, Fruit, and Yield Analysis The results showed that the plants with the compost application started flowering in a shortTerh etimreesu clotsmsphaorweded tot hthate tphleanptlas nwtsitwh ittrheathtme econmt wpiotshtoauptp almicaetniodnmsetnart t(epd-vflaoluwee 0ri.n0g07i)n. a Alsshoo, rptelrantitms ewciothm ipnaorceudlatotiothne wpiltahn tPssweuitdhomtroenaatsm peunttidwai t(hinoduitvaidmueanl damnden cto(p-i-nvoacluuela0te.0d0)7 ). stAarltseod, pfrlaunittisnwg iitnh ain sohcourltaetrio tnimwei tchoPmspeuadroemd otnoa ps lpaunttids aw(iinthd itvhied utraelaatnmdecnot -winiotchuoluatt eidn)osctualrat-ed tiofrnu (ipti-nvgaluine 0a.0s1h2o)r (tFerigtuimree 4)c.o mpared to plants with the treatment without inoculation (p-value 0.012) (Figure 4). Figure 4. Days to flowering in strawberry seedlings cv. San Andreas based on microbial inoculation and organic amendments application. (WM = without microbial inoculation, T = inoculation with Trichoderma sp., P = inoculation with Pseudomonas putida, T + P = co-inoculation, WA = without organic amendments, C = with compost, M = with manure and leaf litter.) Means with different letters in the same factor are statistically different (LSD Fisher, α = 0.05). Appl. Microbiol. 2024, 4, FOR PEER REVIEW 8 Figure 4. Days to flowering in strawberry seedlings cv. San Andreas based on microbial inoculation and organic amendments application. (WM = without microbial inoculation, T = inoculation with Trichoderma sp., P = inoculation with Pseudomonas putida, T + P = co-inoculation, WA = without or- Appl. Microbiol. 2024, 4 ganic amendments, C = with compost, M = with manure and leaf litter.) Means with different letter1s1 17 in the same factor are statistically different (LSD Fisher, α = 0.05). ReRgeagradridnign fgloflwoewres rasnadn fdrufriutsi,t sit, wit awsa osbosbersverevde tdhatht atht eth ceocmopmopsot satpapplipclaitciaotnio tnretaretmatemnetn t increased the number of flowers per m2increased the number of flowers per m (p2 -(vpa-vluaelu 0e.002.70)2,7 w),hwilhei cleo-cion-oincouclautliaotnio inncinrecaresaesde tdheth e nunmubmebr eorfo ffru 2 friutsi tpsepre mr m (2p-(vpa-vluaelu 0e.003.073) 7(F) i(gFuigrue r5e).5 ). FigFuigrue r5e. 5N. uNmumbebre or fo ffrfuruitist saanndd fflloowweerrss ppeerr ssqquuaarreem meeteterri nins tsrtarwawbebreryrrsye esdeelidnlgins gcvs. cSva.n SAann dArenadsrbeaasse d baosendm oincr ombiicarloinboiaclu ilnaoticounlaantidonth aenodr gtahnei coragmaennicd mamenetnsdamppelnictas taiopnp.l(iWcaMtio=n.w (WithMou =t mwiicthroobuiat lminiocrcoublaiatilo n, inoTc=uliantoiocnu,l aTt i=o ninwoictuhlaTtriiochno dweirtmha Tsrpic.,hPod=erimnoa csupl.a, tPio n= iwniotchuPlasetiuodno mwointhas Ppsuetuiddao,mTon+aPs p=uctoid-ain, oTc u+ lPat i=o n, coW-inAoc=uwlaittihoonu, tWoArg a=n wiciathmoeuntd omrgeanntsic, Cam=ewnidthmceonmtsp, oCs t=, Mwi=thw ciothmmpoasntu, rMe a=n wd ilteha fmliattneur.r)eM aenadn slewafi th littdeirf.f)e rMenetalnestt ewrsitihn dthifefesraemnet lfeatctteorrs inint hthees asmame ese fraiecstoarr eins ttahties tsicaamllye dseifrfierse natre(L sStDatiFsitsichaelrl,yα d=if0fe.0r5e)n.t (LSD Fisher, α = 0.05). The fruit’s physical–chemical evaluation results are shown in Table 4. Statistical difTfehre nfrcuesit’ws eprheyosinclayl–fcohuenmdicianl tehvealtuitartaiotanb rlesauclitds iatyre osfhothwenf riun iTtsa,bwleh 4e.r Setathtiesttirceaal tdmife-nt ferweintcheosu wt oerge aonnilcya fmouend mine tnhtes thitorwateadblsel iagchidtliytym oof rtheea cfriduiftrsu, witsh(epr-ev tahlue etr0e.a0t4m7)e.nCt ownictheronuitn g orpgHan,ific rammneensds,mBernixt dshegowreeds, aslnigdhftrluy itmdoimre eancsiido nfsru, itsw (aps-voablus er v0e.0d47th).a Ct tohnecestrundinygf apcHto, rs firmanienstsa,i nBeridx tdhegsraemese, afrnudit fqruiat lditiymaesntshieoncso,n itr wolatsr eoabtsmerevnetsd. that the study factors main- tained the same fruit quality as the control treatments. Table 4. Physical–chemical characteristics and fruit size based on microbial inoculation and organic Taabmlee n4.d Pmheynstiscalp–pchliecmatiocanl. c(hWaMrac=tewriistthicosu atnmdi cfroubiti aslizine obcausleadti on, mTi=crionboicaul liantoiocnulwatitohnT arnicdh oodregrmanaics p., amPe=ndinmoecnutlsa taiopnplwiciathtioPns.e u(WdomMo n=a ws pituhtoiduat, Tm+icPro=bicaol -iinocullattiion,, WT A= i=nwociuthlaotuiotno rwgaitnhi cTarmicheondemrmean ts, spC., P= =w iinthoccuolmatpioons tw, Mith= Pwseiuthdomaonnuasr epuantiddal,e Taf +l iPtt e=r )c.o-inoculation, WA = without organic amend- ments, C = with compost, M = with manure and leaf litter.) pH Treatment pH (g ACicidit Ayc idity SugarsS ugars FirmFniermssn ess Size Treatment tric Acid·100 mL −1) (Brix◦) (kg) Si(zcem 2) (g Citric Acid∙100 mL−1) (Brix°) (kg) (cm2) Interaction n.s. n.s. n.s. n.s. n.s. Interaction n.s. n.s. n.s. n.s. n.s. Fact±or F a1c.t oMr i1c.rMobiciarol biniaolcinuolacu±ti loanti on WM 3.37 0.06 n.s. 0.71 0.06 n.s. 13.33 ± 1.80 n.s. 0.14 ± 0.03 n.s. 1471 ± 107 n.s. WMT 3.37 ± 30..3036± n.0s..1 0 0.71 ± 0.00.766 n±.s.0 .08 13.33 ± 11.82.08 4n.±s. 2.22 0.14 ± 00..1053 ±n.0s..0 3 1471 1±4 15107± n5.5s. T P 3.33 3±.3 08.1±0 0.12 0.76 ±0 0.7.038± 0.09 12.84 ±1 32..6252± 1.83 0.15 0±. 103.0±3 0.03 1415514 1± ±551 1 0 PT + P 3.38 3±.4 00.1±2 0.04 0.73 ±0 0.6.099± 0.04 13.65 ±1 12..873± 2.22 0.13 0±. 106.0±3 0.09 1541413 ±5 1±1017 9 T + P 3.40F a±c t0o.r024. Organic am0en.6d9m ±e 0n.t0s4a pplication 12.7 ± 2.22 0.16 ± 0.09 1435 ± 179 WA Facto3r .323. O±r0g.a09ninc. sa.mendment0s. 7a6p±pl0ic.0a7tiaon 12.99 ± 1.77 n.s. 0.14 ± 0.03 n.s. 1492 ± 169 n.s. C abWA 3.33 ± 30..3099± n.0s..0 6 0.76 ±0 .07.10±7 a0 .06 b 12.99 ± 1 1.73.73 3n.±s. 2.12 0.14 ± 00..1043 ±n.0s..0 4 1492 1±4 18569± n8.8s. C M 3.39 3±.3 09.0±6 0.09 0.71 ± 00.6.096± 0 .07 13.33 ±1 32..0182± 2.17 0.14 0±. 104.0±4 0.08 1418454 7± ± 102ab 88 M 3.39 ± 0.09 Means with d0if.f6e9re n±t 0le.t0t7er sb in the same f1ac3t.o0r8in ±t h2e.1s7am e column a0r.e1s4t a±ti s0ti.c0a8ll y different (L1S4D47F i±sh 1er, α = 0.05);n.s.: no significance. 02 In terms of crop yield, the results showed that the organic amendments application, with either compost or manure, significantly increased the grams of fruit per plant (p-value 0.023). In the microbial inoculation cases, we did not find significant differences (Table 5). Appl. Microbiol. 2024, 4, FOR PEER REVIEW 9 Means with different letters in the same factor in the same column are statistically different (LSD Fisher, α = 0.05); n.s.: no significance. In terms of crop yield, the results showed that the organic amendments application, with either compost or manure, significantly increased the grams of fruit per plant (p- value 0.023). In the microbial inoculation cases, we did not find significant differences Appl. Microbiol. 2024, 4 (Table 5). 1118 Table 5. Strawberry crop yield cv. San Andreas based on microbial inoculation and the organic Taambelend5m. Sentrta awpbpelircrayticorno.p (WyiMeld = wcvi.thSoaunt Amnicdrroebaisalb iansoecduloantiomni,c Tr o=b iinaol ciunloactuiolnat wiointha Tnrdicthhoedeorrmga nspic., aPm =e inndomcuelnattiaopnp wlicitaht iPosne.u(dWomMon=asw piuthtioduat, Tm +ic Pro =b ciaol-iinnooccuullaattiioonn,, WT =Ai =n owciutlhaotiuotn owrgiathniTcr aicmhoednedrmaenspts.,, PC= = iwnoitchu lcaotmionpowsti,t hMP =se wudiothm monaans upureti adna,dT le+aPf l=ittceor-.i)n oculation, WA = without organic amendments, C = with compost, M = witFhrmuiatn Wureeiagnhdt leaf litter). Yield Treatment (gF Fruruitit−1We) ight (g Fruit Plant−1)Y ield (t ha−1) IntTerreaactmtioenn t (ng.sF.r uit−1) (g FruitnP.lsa. nt−1) (t han−.1s). Interaction Fanct.so.r 1. Microbial inocunla.st.ion n.s. WM 21.29 ± 1F.a1c5t onr.s1.. Microbial in1o7c3u.6la ±ti o6n5.3 n.s. 9.3 ± 1.1 n.s. TW M 202.16.219 ±± 1.12.135 n.s. 1732.612±.96 ±5. 345n..7s . 9.3 ±9.18.1 ±n 1.s.5. P T 20.9260. 6±1 1±.891 .23 21823.9.7± ±4 753.7.7 9.88±.9 1±. 53.0 T + PP 20.3260. 9±6 1±.321 .89 128031.7.2± ±7 834.7.3 8.910±.33 ±.0 2.0 T + P Fact2o0r. 326. ±Or1g.3a2nic amendm2e0n1t.2 a±pp8l4i.c3ation 10.3 ± 2.0 WA 20.9F1ac ±to 1r.26.0O nr.gsa. nic amendme1n5t2a.p7p ±l i7ca8t.i0onbb 9.3 ± 2.7 n.s. CW A 20.9C 21.120 1 ±± 1.60 n.s. 152.7 ± 78.0 a 9.3 ± 2.7 n.s.1.10 1±.521 .52 22114.44.4± ±4 488.8.8a 9.99±.9 1±. 61.6 MM 20.4210. 4±1 1±.091 .09 22111.14.4± ±5 588.4.4a a 9.69±.6 1±. 71.7 Meeaanns sw withitdhi fdfeirfefnetrelenttte lrestitnerths einsa mtheef ascatmorei nfathcteosra mine tchoelu smanmaer ecsotalutimstinca allryed siftfaetriesnttic(aLlSlDy Fdiisfhfeerr,eαnt= (0L.0S5D); nF.sis.:hneor,s iαg n=i fi0c.0a5n)c;e n. .s.: no significance. FFiiggurreess 66 aannd 77 sshhoow tthhee Prriinncciipaall Coomppoonneenntt AAnnaalylyssisis (P(PCCAA) )fofor rmmicircorobbiaila lininocouc-- ulalatitoionn anandd thteh eapaplpiclaictaiotino onfo ofrgoargnaicn aicmaemndenmdemntesn, trse,srpeescpteivcetilvye. lIyn. bIontbho ctahsecsa,s tehse, ftihrestfi twrsot tpwroinpcirpinacl icpoaml cpoomnepnotns eenxtpslaeixnp olavienr o8v0e%r 8o0f %theo ftotthael vtoatrailanvcaer.i aTnhcee .saTmhpelseas mthpalte asrteh oant athree opnostihtievpe oasxiitsiv oef aPxCis1 ohfaPveC 1a ghraevaetear ginreflautenr cien floune mncoesto onf mthoes vt aorfiathbelevs aervialbuleasteedv (aFliugautreeds (6Fbig aunrdes 76bb).a Ondn 7tbh)i.s Obansitsh, iws eb aosbis,erwvedo bthseart vceod-inthoactuclaot-iionno c(Fuilgautiroen 6(aF)i gaunrde t6hae) caonmdptohset caopmppliocastiaopnp (lFicigatuioren 7(aF)i gaurere th7ae) tarereattmheentrtesa tthmaet nhtasdth tahteh baedstt hresbueltsst irnes tuhlets vianritahbelvesa.r iables. (a) (b) FFiigguurree 66.. PPrriinncciippaall CCoommppoonneenntt AAnnaallyyssiiss ((PPCCAA)) sshhoowwiinngg tthhee iinntteerrrreellaattiioonn ooff eevvaalluuaatteedd ppaarraammeetteerrss in strawberries according to microbial inoculation. (a) Distribution of inoculation treatments in the in strawberries according to microbial inoculation. (a) Distribution of inoculation treatments in the principal components (Black = Without inoculation; Aqua = Inoculation with Trichoderma sp.; Purple: Inoculation with Pseudomonas putida; Green: Co-inoculation). (b) Distribution of the variables in the principal components (NL = Number of leaves; H = Height; LA = Leaf area; FW_a = Fresh weight aerial; FW_r = Root fresh weight; DW_a = Dry weight aerial; DW_r = Root dry weight; DtF = Days to fruiting; Fl = Number of flowers; Fr = Number or fruits; W_fr = Fruit weight; Y_gr = Yield expressed in grams of fruit per plant; Y_tn = Yield expressed in tons per hectare; pH = pH; S = Sugars; Ac = Acidity; Fir = Firmness). Appl. Microbiol. 2024, 4, FOR PEER REVIEW 10 principal components (Black = Without inoculation; Aqua = Inoculation with Trichoderma sp.; Pur- ple: Inoculation with Pseudomonas putida; Green: Co-inoculation). (b) Distribution of the variables in the principal components (NL = Number of leaves; H = Height; LA = Leaf area; FW_a = Fresh weight aerial; FW_r = Root fresh weight; DW_a = Dry weight aerial; DW_r = Root dry weight; DtF = Days to fruiting; Fl = Number of flowers; Fr = Number or fruits; W_fr = Fruit weight; Y_gr = Yield ex- Appl. Microbiol. 2024, 4 pressed in grams of fruit per plant; Y_tn = Yield expressed in tons per hectare; pH = pH; S = Sug1a1r1s;9 Ac = Acidity; Fir = Firmness). (a) (b) FFiigguurree 77.. PPrriinncciippaall CCoommppoonneenntt AAnnaallyyssiiss ((PPCCAA)) sshhoowwiinngg tthhee iinntteerrrreellaattiioonn ooff eevvaalluuaatteedd ppaarraammeetteerrss in strawberries according to the application of organic amendments. (a) Distribution of inoculation tirneasttmraewnbtse rirni etshea cpcroirndciinpgalt ocotmhepaopnpenlitcsa t(iBolnacokf: oWrgiathnoicuat moregnadnmic eanmtse.n(da)mDeinstt;r iAbuqutiao:n woifthin coocmulpatoisotn; Gtrreeaetnm: ewnittshi nmtahneuprrei nacnidp alleacof mlitpteorn).e n(bts) (DBilsatcrkib:uWtiiotnh oouft tohreg avnariciaabmleesn idnm theen tp; rAinqcuipa:alw ciothmcpoomnpenotsst ; (GNrLe e=n N: wumithbemr oafn lueraevaens;d Hl e=a Hf leititgehrt);. L(bA) =D Lisetarfi bauretiao; nFWof_tah =e Fvraersiahb wleesiginhtt haeerpiarli;n FcWip_arl c=o Rmopoot nfreenshts w(NeLig=htN; DuWmb_ear =o Df lreya wveesi;gHht= aeHreiaigl;h Dt;WLA_r == LReoaofta drerya; wFWeig_hat=; DFtrFes =h Dwaeyigs htot afreuriiatiln; FgW; F_l r= =NRuomobt efrre oshf fwloewigehrst; DFrW =_ Na u=mDbreyrw oer ifgrhutitase; rWia_l;fDr =W F_rru=it Rwoeoitgdhtr;y Yw_geirg =h tY; iDeltdF =exDpareysssetod firnu igtirnagm;sF lo=f fNruuimt pbeerr polfaflnot;w Ye_rtsn; =F rY=ieNldu emxpbreersoserdfr iuni ttso;nWs _pferr= heFcrtuairtew; peHig h=t p; YH_; gSr == SYuigealdrse; xApcr e=s Asecdidiintyg; rFaimr =s Foifrfmrunietspse).r plant; Y_tn = Yield expressed in tons per hectare; pH = pH; S = Sugars; Ac = Acidity; Fir = Firmness). 4. Discussion 4. DiTsrciuchsosdioernma sp. inoculation and the co-inoculation of Trichoderma sp. with Pseudomo- nas puTtriidcah owdeerrme afosupn. din tooc ublea ttihoen traenadtmtheenctos -winiothcu tlhaet iloonwoefsTt rmicohortdaelrimtya psper.cwenitthagPes,e luedsosm thonaans 7p%ut.i dSaimwilearre rfeosuunltds htoavbee bteheent rreeaptomrteendt swwitihth ththe eaplopwliceasttiomno ortfa eliftfyecptievrec emnticargoeo,rlgeasns itshmasn s7u%p.pSleimmielnatrerde swulitths hbaovkeasbheie, nwrheperoer tsetdrawwibthertrhiees arpepaclihceadti olensso fthefafne cati v10e%m imcroorotargliatyn irsamtes [s2u2p].p Ilne motehnetre dstwuditihesb, omkoasreh it,hwanh e9r8e%st roafw stbrearwribeesrrreya csheeeddlliensgsst ihnaoncua l1a0t%edm woirthta Tlitryichraotdeer[m22a] . sIpn. o[t2h3e]r wsteurde iaebs,lem toor aecthhiaenve9 8r%oootifnsgtr. aHwobwerervyesre, ePdsleiundgosminonoacsu lpautetiddaw hiaths Tar hicihgohdeerr mmaosrpta. l[i2t3y] rwateer,e siamblielatro toa cthhiee uvneirnoooctuinlagt.edH torweaetmveern, tP. seudomonas putida has a higher mortality rate, similTahret odtihffeeruennicneo cbueltawteedent rTearitcmhoednetr.ma sp. and Pseudomonas putida is likely due to the greateTrh eeffdicifafceyre innc ecobnettrwoleleinngT rroicohto ddeirsmeaasessp .anandd TPriscehuoddoemrmoan asspp. uctaiudsainisgl igkreelaytedru reotoot tbhi-e ogsrteimateurlaetfifiocna. cRyoiont cpoantthrooglleinngs rsouocthd aiss ePahsyetsoapnhtdhoTrraic shpo.d, eFrmusaarsipu.mc asups.,i nagndgr Reahtiezrocrtooontiab siops. - atrime tuhlea tmioani.nR loimotitpinatgh foagcetonrsss iunc hstraaswPbheyrtroyph etshtoarbalsisph.,mFeunsta r[i2u4m]. sTph.e, saen dcaRuhsiez orcottotniniag sopf. tahree ctrhoewmna ainndlim laitteinrgalf aroctootrss. Tinhset rmawecbhearnryisemstsa bulsieshdm beyn Tt r[i2c4h]o.dTehrmesae scpa.u tsoe crootnttinrogl orfotohte pcartohwon- gaennds laartee raanl trioboiotss.isT, hmeymcoepcharaansiistmisms u, isneddubcyedT rriechsiosdtearnmcea,s apn.dto ncicohnetrol root pathogens areantibiosis, mycoparasitism, induced resistance, and niche exclusion [ e2x5c].luPsrieovnio [u2s5]r.e Pseraervcih- osuhso wressetharacthg slyhootwoxs itnhsa,tv girlyidoitno,xtirnisc,h voidriedrimn,i ntr,iacnhdodfeurrmanino,n aenadr efuthraenmonaein amree tthaeb moliatiens mthea-t tdaebgorliatdese tthhaetc deellgwraadllea tnhde ccaelul swe aplal tahnodg ecnauicshe yppahthaoe-gpernoigc rhaympmhaeed-pcerollgdreaamthm[e2d6 ]c.ell death [26]. Strawberry’s high sensitivity to salt stress could cause the low rotting percentage [27] obtaSintreadwibnetrhryis’sr ehsigeahr scehn(stihtievimtya txoi msaultm strveaslsu ceowulads c9a3u%se) .thOes lmowot ricotatinndg opxeirdcaentitvaegest [r2e7s]s ocbatuasiendedb yina thhiigsh relesveealrcohf s(athltes imnhaxibimitsusmtr avwalbueer rwyarso o9t3d%e)v. eOlospmmoetinct ainndth oexeidaraltyivdee vsterleosps - ment stages [28]. Trichoderma sp. promotes root growth by releasing auxin intermediary metabolites such as indole 3-acetaldehyde acid (IAA) and indole 3-ethanol (IET) [29]. Fur- thermore, under salt stress conditions, Trichoderma sp. produces 6-pentyl-α-pyrone (6-PP), a volatile organic compound that inhibits primary root growth and induces lateral root growth [30]. However, to determine the influence of Trichoderma sp. on the mortality rate, more detailed studies should be carried out. Regarding vegetative growth, it was found that the combination of Trichoderma sp. with compost resulted in the highest values for leaf number and plant height during the Appl. Microbiol. 2024, 4 1120 early growth stages. However, at 90 dat, only the compost explained the highest number of leaves and the co-inoculation of Trichoderma sp. and Pseudomonas putida explained the highest leaf area. Also, microbial co-inoculation and the compost application increased the aerial dry biomass. Increased biomass expressed as greater leaf number and plant height is related to a major leaf photosynthetic activity [31]. Sucrolytic activity in Trichoderma sp. fungal cells, mediated by intracellular invertase, is known to direct the systemic induction of photosynthesis and sucrose partitioning to the roots [32]. In addition, Trichoderma sp. causes plant gene expression reprogramming to induce the rubisco activity and the oxygen-generating complex of the photosystem II [33]. The leaf area increase is likely due to an increased requirement for photosynthetic activity. In addition, synergistic action between T. harzianum and P. fluorescens in increasing nitrogen and phosphorus uptake has been reported, resulting in higher aerial biomass [34]. Both Pseudomonas sp. and Trichoderma sp. can exert beneficial and biocontrol effects on plants; however, studies show that combinations of the two are more effective than each individually [35,36]. This situation was observed in our results, as co-inoculated seedlings showed greater leaf area and aerial biomass, statistically different from those in which only one microorganism was inoculated. In addition, compost use as an organic amendment or substrate for Trichoderma sp. development resulted in greater plant growth stimulation. This positive effect of compost agrees with other studies, where the incorporation of organic amendments in strawberry cultivation increases the number of leaves [37]. The positive effect of co-inoculation could be associated with synergism between bioactive molecules or volatile organic compounds of both strains [38]. Biomass increase due to the integrated effect of different rhizospheric strains has been reported in strawberry cultivation [39]. Also, this response could be attributed to chlorophyll content increase, an indirect indicator of leaf photosynthetic activity [40]. This study revealed the positive effect of the interaction between compost and co-inoculation on biomass increase. There is scientific evidence justifying the increased nitrogen mineralization and phosphatase activity in the soil due to the inoculation effect of Trichoderma sp. supplemented with compost [41]. Also, studies indicate that the application of compost improves microbial activity and thus generates the production of growth regulators by microorganisms, which is reflected in an increase in biomass in the plant. [42]. This is supported by other studies with strawberry seedlings grown under greenhouse conditions, where it is shown that compost helps Trichoderma sp. populations to remain stable in the soil [43]. Inoculation with Pseudomonas putida (individual or co-inoculation with Trichoderma sp.) influenced the days to flowering, reducing the time by an average of 4 days. These results have been rarely reported in fruit crops; however, it has been found in the literature that Pseudomonas sp. influences flowering. In a study on the chrysanthemum, Pseudomonas sp. accelerated flowering by an average of 14 days compared to the control [44]. Another study on the chrysanthemum showed that two strains of Pseudomonas putida, characterized as indole acetic acid (IAA) producers, improved flowering [45]. So, it is likely that this influence is related to the improvement in nutrient absorption and the transport of growth-promoting substances, resulting in an early transformation of plant parts from the vegetative to the reproductive phase. On the other hand, organic amendments not only provide nutrients to the plant but also contribute organic matter, thereby increasing soil fertility [46]. The addition of compost is known to stimulate microbial growth due to the increased amount of organic carbon it provides [47]. It can also have a direct effect on the plant due to the microorganisms present in it. Manure improves soil’s chemical and physical properties such as soil water, bulk density, erosion resistance, and nutrient status, among others, which can favor crop growth and productivity [48]. The benefits of the amendments application were reflected in the yield increase (g fruit plant−1) observed in our results, which correlates with what is found in the literature. Some studies conclude that the addition of vermicompost improved growth and yield levels in strawberries [49,50]. Also, significant differences in strawberry yield and fruit quality were found with the addition of chicken manure [51]. Appl. Microbiol. 2024, 4 1121 5. Conclusions Pseudomonas putida inoculation accelerated the seedling’s flowering process and co- inoculation (Trichoderma sp. + Pseudomonas putida) increased the leaf area, fresh biomass, and dry biomass, both aerial and root, and the number of fruits per square meter. Concerning the organic amendments, only the compost treatment stood out, with higher values found in the number of leaves, the aerial dry biomass, the days to flowering, the number of flowers per square meter, and the fruit yield per plant. Under the conditions in which the experiments were carried out, these results show that compost application at 15 t·ha−1 and co-inoculation of Trichoderma sp. and Pseudomonas putida have potential as organic technologies in Fragaria sp. cultivation; however, it is important to repeat these treatments in other seasons and regions to validate the effect observed in our study. Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/applmicrobiol4030075/s1, Table S1: Aerial and root dry biomass at 90 dat based on the interactions of the two study factors. Author Contributions: Conceptualization, H.C.-S. and R.S.; formal analysis, L.H.; investigation, L.H. and T.S.; methodology, L.H., T.C., L.A. and T.S.; project administration, R.S.; supervision, R.S.; writing—original draft, L.H.; writing—review and editing, R.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the INIA project “Mejoramiento de los servicios de inves- tigación y transferencia tecnológica en el manejo y recuperación de suelos agrícolas degradados y aguas para riego en la pequeña y mediana agricultura en los departamentos de Lima, Áncash, San Martín, Cajamarca, Lambayeque, Junín, Ayacucho, Arequipa, Puno y Ucayali” CUI 2487112. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors appreciate the participation and logistical support of Pedro Nicho and José Cóndor Caro, research specialists in the EEA Donoso INIA. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Fernández-Lara, R.; Gordillo, B.; Rodríguez-Pulido, F.J.; Lourdes González-Miret, M.; del Villar-Martínez, A.A.; Dávila-Ortiz, G.; Heredia, F.J. 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