microorganisms
Article
Effect of Co-Inoculation with Growth-Promoting Bacteria and
Arbuscular Mycorrhizae on Growth of Persea americana
Seedlings Infected with Phytophthora cinnamomi
Richard Solórzano-Acosta 1,2,*, Marcia Toro 2,3 and Doris Zúñiga-Dávila 2,*
1 Centro Experimental La Molina, Dirección de Supervisión y Monitoreo en las Estaciones Experimentales
Agrarias, Instituto Nacional de Innovación Agraria (INIA), Av. La Molina N◦ 1981, Lima 15024, Peru
2 Laboratorio de Ecología Microbiana y Biotecnología, Departamento de Biología, Facultad de Ciencias,
Universidad Nacional Agraria La Molina, Lima 15024, Peru; mtoro@lamolina.edu.pe
3 Centro de Ecología Aplicada, Instituto de Zoología y Ecología Tropical, Facultad de Ciencias,
Universidad Central de Venezuela, Caracas 1041-A, Venezuela
* Correspondence: investigacion_labsaf@inia.gob.pe (R.S.-A.); dzuniga@lamolina.edu.pe (D.Z.-D.)
Abstract: Avocado is one of the most in-demand fruits worldwide and the trend towards its sustain-
able production, regulated by international standards, is increasing. One of the most economically
important diseases is root rot, caused by Phythopthora cinnamomi. Regarding this problem, antago-
nistic microorganism use is an interesting alternative due to their phytopathogen control efficiency.
Therefore, the interaction of arbuscular mycorrhizal fungi of the phylum Glomeromycota, native
to the Peruvian coast (GWI) and jungle (GFI), and avocado rhizospheric bacteria, Bacillus subtilis
and Pseudomonas putida, was evaluated in terms of their biocontrol capacity against P. cinnamomi
in the “Zutano” variety of avocado plants. The results showed that the GWI and Bacillus subtilis
combination increased the root exploration surface by 466.36%. P. putida increased aerial biomass by
360.44% and B. subtilis increased root biomass by 433.85%. Likewise, P. putida rhizobacteria showed
the highest nitrogen (24.60 mg · g−1 DM) and sulfur (2.60 mg · g−1 DM) concentrations at a foliar
level. The combination of GWI and Bacillus subtilis was the treatment that presented the highest cal-
Citation: Solórzano-Acosta, R.; Toro,
cium (16.00 mg · g−1 DM) and magnesium (8.80 mg · g−1 DM) concentrations. The microorganisms’
M.; Zúñiga-Dávila, D. Effect of
multifunctionality reduced disease severity by 85 to 90% due to the interaction between mycorrhizae
Co-Inoculation with Growth-
and rhizobacteria. In conclusion, the use of growth promoting microorganisms that are antagonistic
Promoting Bacteria and Arbuscular
Mycorrhizae on Growth of Persea to P. cinnamomi represents a potential strategy for sustainable management of avocado cultivation.
americana Seedlings Infected with
Phytophthora cinnamomi. Keywords: Bacillus; Pseudomonas; PGPB; antagonists; root rot
Microorganisms 2024, 12, 721.
https://doi.org/10.3390/
microorganisms12040721
1. Introduction
Academic Editor: Giovanni Vallini
Avocado is one of the most popular tropical fruits in the world due to its high nu-
Received: 25 February 2024 tritional value and pleasant flavor [1]. Various problems affect the avocado crop, which
Revised: 18 March 2024 reduce its yield and, therefore, result in lower income for producers [2] due to poor soil
Accepted: 29 March 2024 and water resource management that lead to root rot disease [3].
Published: 2 April 2024 Root rot caused by Phytophthora cinnamomi is the most destructive avocado disease
worldwide [4]. Primary symptoms are shown in the roots, which take on a dark brown
color, become brittle, are easily detached, lose salt selectivity, and get infected by vascular
Copyright: © 2024 by the authors. pathogens. Secondary symptoms begin with leaves yellowing, progressive defoliation, and
Licensee MDPI, Basel, Switzerland. general decay (epinasty), similar to water stress, despite having soil in optimal humidity
This article is an open access article conditions [5].
distributed under the terms and Available control methods can reduce this disease’s severity [6]. However, the current
conditions of the Creative Commons trend is to incorporate new technologies that reduce agrochemical use to obtain healthier
Attribution (CC BY) license (https:// products and, at the same time, open more demanding markets [7]. In this sense, rhizobacte-
creativecommons.org/licenses/by/ ria and mycorrhizae constitute important sources of potentially beneficial microorganisms
4.0/). with plant growth-promoting activity and antagonistic effects against phytopathogens [8,9].
Microorganisms 2024, 12, 721. https://doi.org/10.3390/microorganisms12040721 https://www.mdpi.com/journal/microorganisms
Microorganisms 2024, 12, 721 2 of 14
Arbuscular mycorrhiza-forming fungi (AMF) belonging to the Glomeromycota phy-
lum are a crucial part of rhizospheric microbial communities [10]. These microorganisms
establish symbiotic relationships with plants, playing an essential role in their nutrition and
development. Moreover, they offer additional benefits such as increasing plants’ tolerance
to biotic stresses like phytopathogen control. As a result, the commercial use and mass
production of these fungi have generated significant interest [11].
Mycorrhizae are known to enhance the resistance of plants to pathogen attacks [12],
particularly those that affect the root. When fungi establish themselves before pathogens,
they reduce the incidence of damage. The benefits of mycorrhizae include an increase in
root system biomass and nutrient absorption, which is facilitated by the fungal external
mycelium [13]. Additionally, mycorrhizal plants activate the expression of genes that
are related to resistance to pathogens. This may be attributed to other mechanisms that
mycorrhizae activate in plants [14].
Recent research indicates that rhizobacteria, which are naturally found in soils, have
the potential to control the harmful effects of P. cinnamomi-related diseases [15,16]. The
Bacillus and Pseudomonas strains are effective as biofertilizers and biocontrol agents in
agriculture. These bacteria can suppress pathogenic microbes, promote plant growth, and
facilitate the assimilation of nutrients, resulting in beneficial effects [17,18].
For these reasons, this study of interactions between growth-promoting microorgan-
isms will allow for overcoming the negative effect of root rot caused by P. cinnamomi on
avocado crops. In this sense, this research’s main objective is to evaluate the effects of Phy-
lum Glomeromycota mycorrhizae and B. subtilis and P. putida rhizobacteria co-inoculation
on severity reduction of P. cinnamomi in “Zutano” variety avocado plants. Likewise, this
research aims to identify the response in terms of vegetative and root growth promotion
in plants infested with the disease. Finally, it seeks to relate co-inoculation, plant growth
response, and macronutrient accumulation at a foliar level.
2. Materials and Methods
2.1. Bacteria–Mycorrhiza Co-Inoculation Trial Design
A complete randomized design (CRD) with factorial arrangement was used in this
experiment. Two factors, each with three levels, were tested. The factors were mycorrhizae
(without mycorrhizae, coastal mycorrhizal native Glomeromycota fungi from a wetland
(GWI), and jungle mycorrhizal native Glomeromycota fungi from a fallow field (GFI)) and
PGP bacteria with antagonistic activity in vitro on P. cinnamomi (without bacteria, B. subtilis
Bac F, and P. putida P3). The experiment generated a total of nine treatments, each with six
replicates, and one plant per replicate. For this trial, the seeds were first germinated, then
inoculated with bacterial promoter, and, after a week, with mycorrhizae, depending on the
treatments. The inoculations were allowed to be established for 21 days after inoculation
with mycorrhizae before the plants were exposed to the phytopathogen P. cinnamomi. Seed
germination and a description of the substrate used for the trials are discussed below.
Premix® Nº8 substrate was used (pH: 5.5, EC: 0.75 dS · m−1, P: 25 ppm, and K:
100 ppm), which was moistened to field capacity and sterilized in an autoclave at 121 ◦C
and 15 lbs pressure. The sterile substrate was added to 2 L plastic pots. One avocado var.
Zutano seed was planted per pot, weighing approximately 50 g, which was irrigated with
100 mL of sterile water. The seeds, donated by the Avo Hass Perú company, were previously
sterilized in 10% sodium hypochlorite for 10 min, and once rinsed, they were sown and
allowed to germinate for ten days in a greenhouse with 20 ◦C average temperature and
60% humidity until seedlings were obtained before treatment with the inoculants.
2.2. Inoculating Seedlings with Bacterial Strains
The bacteria used in this research were isolated from the avocado rhizosphere and
were selected for their ability to grow in saline conditions in vitro. This was the subject of
another publication in which their effectiveness in attenuating salinity was tested. Strains
were cultured in nutrient broth for 48 h at 28 ◦C. Then, 30 mL of the bacterial broth from
Microorganisms 2024, 12, 721 3 of 14
each strain, with a 108 CFU · mL−1 concentration, was applied to the plant neck. After
15 days of germination, this was performed in the rootlet’s influence area. This procedure
was repeated a second time, 3 days after the first inoculation, to ensure that the bacteria
were present and suitable for the plant rhizosphere before starting the treatments.
2.3. Arbuscular Mycorrhiza Selection, Propagation, and Inoculation
Two Glomeromycota fungi inocula isolated from Peru were used. One was obtained
from rhizosphere soil from a fallow land in Pucallpa, Ucayali (Glomeromycota fallow
inoculum—GFI), and the other from rhizosphere soil from Sporobolus sp. located in Pisco,
Ica (Glomeromycota wetland inoculum—GWI), according to Castañeda et al. [19]. Both were
reproduced in trap pots with Brachiaria decumbens in sterile sand, and watered with Long
Ashton’s solution and a quarter dose of phosphorus (P) every 15 days [20].
Rhizosphere samples were taken to quantify spores and arbuscular mycorrhizae (AM)
colonization of rootlets [21] in each inoculum. Similar spore numbers were obtained in GFI
and GWI to inoculate avocado plants in the experiment. The GFI inoculum consisted of
native species, mainly R. intraradices fungus and Acaulospora, Gigaspora, and Archaeospora
genera, of which 3726 spores per mL per seedling were added from colonized rootlets
(70%). In the GWI inoculum, R. intraradices fungus predominated, of which 3400 spores per
mL per seedling were applied from colonized rootlets (90%). In both cases, inocula were
applied to the base of the germinated seeds, then spread around, and the substrate was
uncovered slightly with a sterile spatula to expose the rootlets without hurting them.
2.4. Propagation and Inoculation of P. cinnamomi
Propagation was carried out on carnation petals, which were previously disinfected
in 1% sodium hypochlorite for 60 s, and then rinsed with sterile water. Petals were
placed in a 250 mL sterile flask with 100 mL of sterile mineral water. Then, agar discs
containing P. cinnamomi were placed on the petals, allowing the pathogen to colonize
them. Afterward, inoculation was performed (Figure 1). Infection of seedlings with P.
cinnamomi was carried out by applying 100 mL of zoospore solution at 105 zoospores · mL−1
concentration around the avocado seedling neck on two occasions spaced one week apart.
The P. cinnamomi strain was identified by the Laboratorio Agrícola Biaster SAC company
in Ica. Pathogen inoculation was carried out two weeks after the last inoculation with
mycorrhizae to promote their establishment. It should be mentioned that prior inoculation
is crucial because, as this is an irreversible disease, a preventive or mitigating effect is
sought before infection.
2.5. Greenhouse Environmental Conditions
During the experiment, the highest temperatures recorded ranged from 24.51 to
27.43 ◦C, while the lowest temperatures ranged from 18.91 to 20.37 ◦C. The relative humid-
ity varied between 59.9 and 61.92%. These measurements were taken by the meteorological
station situated inside the greenhouse of the Microbial Ecology and Biotechnology Labora-
tory at the National Agrarian University La Molina.
2.6. Determination of Harvest, Growth, and Nutrition Characteristics
The experiment lasted for 20 weeks and involved growing plants from germination
to harvesting for nutrient analysis. The germination process took 2 weeks, followed by
inoculation and establishment of PGP bacteria and arbuscular mycorrhizae for 4 weeks,
infection with the pathogen for 2 weeks, and finally, plant growth for 12 weeks. After
this time, various characteristics of plant growth, such as plant height (cm), root length
(cm), and number of leaves, were measured. Fresh and dry aerial biomass (g), fresh and
dry root biomass (g), root moisture (g), and root length/root dry weight ratio were also
evaluated as an index of response to the pathogen that causes root damage. The dry
biomass was determined by drying aerial and root parts in an oven for two weeks at 40 ◦C.
Microorganisms 2024, 12, 721 4 of 14
Microorganisms 2024, 12, x FOR PEER REVIEW 4 of 16 
 Root humidity was calculated by finding the difference between fresh and dry biomass,
which indicates the root tissue water content and root health state.
a 
b c 
 
FFiigguurere1 .1(. a()aP) .Pc.i ncninanmaommoi minio icnuolucmulupmrep parreaptioanraitniomni niner maliwneartearl awnadtecar rannadti ocnarpneatatilso;n(b p)eztoaolss;p (obre) szoospores 
((11000000××));;( (cc))m myycecleiulimuman adnsdp osrpaonrgainagoina coanrn caatironnaptieotnal sp.etals. 
2.7. P. cinnamomi Infection Severity
2.5. GThreeensehvoeursitey EonfviPr.ocninmneanmtaolm Ci oinnfdeictitoionns in avocado seedlings that were previously
inoculDatuedriwngit hthPes euxdpoemroinmasenspt,. tahned hBiagchilleusst stpe.mstpraeirnastuwraessd retceormrdineedd rbaynguesidn gfrtohme v 2is4u.5a1l  to 27.43 
s°cCal,e wphroilpeo sthede bloywReasmt ítreemz apnedraMtuorreasle sra(n20g2e0d). fTrhoems c1a8le.9i1s btoas 2ed0.3o7n e°xCt.e rTnhael  sryemlaptitvome hs umidity 
avnadriwedas bveatlwideaetend 5w9.i9th atnhde  i6n1o.c9u2l%um. Tahmeosuen mt teharosuurgehmreegnrtess swioenrea ntaalkyesins .by the meteorological 
2s.t8a.tSiotanti sstiitcuaal tAenda liynssiside the greenhouse of the Microbial Ecology and Biotechnology Labor-
atorTyh aet stthaeti sNticaatlioannaally Asigs roafrtihaene Uxpneirvimeresnittyw Lasa cMonodluincate.d using IBM’s Statistic Package
for Social Sciences (SPSS) program, version 26. Data collected in each experiment were ana-
l2y.z6e.d Duestienrgmaincoamtiopnle otef Hraanrdvoemsti,z Gedrodwetshig, nan(CdR NDu)twriittihonfa Ccthoariraalcatrerraisntgicesm ent through test F.
The teTstheed efaxcptoer’ismmeanitn leafsfetecdts fwoerr 2e0c awlceuelaktse da,nadn dininvoclavsedo fgsriognwifiincagn pcela, nthtes tfrreoamtm genetrsmination 
wtoe rheacrovmepstairnegd ufosirn gnuDturinecnatn ’asnteasltytsoisd. eTtehrem gineermdififnearetinocnes pbreotwceesesn tboaockte r2ia wl setreakins,s faonldlowed by 
minyoccourrlhaitzioaen. aAnpdr oebsatabbilliitsyhomf aelnpth oafe rPrGorPo fblaecstsetrhiaan a5n%d warabsucoscnusildaerr medysciognrrifihcizanaet. for 4 weeks, 
infection with the pathogen for 2 weeks, and finally, plant growth for 12 weeks. After this 
time, various characteristics of plant growth, such as plant height (cm), root length (cm), 
and number of leaves, were measured. Fresh and dry aerial biomass (g), fresh and dry 
root biomass (g), root moisture (g), and root length/root dry weight ratio were also evalu-
ated as an index of response to the pathogen that causes root damage. The dry biomass 
was determined by drying aerial and root parts in an oven for two weeks at 40 °C. Root 
humidity was calculated by finding the difference between fresh and dry biomass, which 
indicates the root tissue water content and root health state. 
 
Microorganisms 2024, 12, 721 5 of 14
3. Results
3.1. Effect of Bacteria–Mycorrhiza Co-Inoculation on P. Americana var. Zutano Seedlings’ Growth
Parameters after Infection with P. cinnamomi
Mycorrhizae show general activity affecting avocado root length and the relationship
between root length and root dry weight, which results in greater root efficiency by gener-
ating a greater exploration surface concerning the biomass generated (Figures 2–4). They
also reduce the severity of the disease (Table 1, Figure 5). In all cases, the presence of myc-
orrhizae in the roots was verified (Figure 6). Additionally, B. subtilis and P. putida bacteria
promote plant growth by positively affecting various growth parameters such as plant
height, root length, number of leaves, fresh and dry biomass, and root humidity. B. subtilis
stands out for its positive impact on root humidity (Table 2). However, co-inoculating
mycorrhizae with antagonistic bacteria such as P. putida and B. subtilis can decrease plant
height (Figure 2). There is a positive synergistic effect when coastal mycorrhizae and B.
subtilis are combined, promoting root length (Figure 3). The same is seen with jungle
mycorrhizae and B. subtilis for the number of leaves. Inoculating P. putida favors aerial
Mbiicrooomrganaissmss 2p02r4o, 1d2, ux FcOtRi oPEnER ,  RwEVhIEiWle B. subtilis favors overall avocado seedling growth, es7p oef c16i ally
Mthicreooragacncisumsm 202u4,l 1a2t, xio FOnR oPEfEaR eRErViaIElWa nd root biomass (Table 2). 7 of 16 
 
100.00
90.00
100.00
80.00 d d
90.00
70.00
cd cd80.00
60.00 bc d
d
70.00 abc abc
50.00 cd
cd
ab
60.00 a bc
40.00 abc abc
50.00 ab
30.00 a
40.00
20.00
30.00
10.00
20.00
0.00 GWI+B. 
10.00 GWI+P. GFI+B. GFI+P. Only GWI Only GFI B. subtilis P. putida
Without 
subtilis putida subtilis putida bacteria and 
0.00 GWI+B. GWI+P. GFI+B. TreGaFtI+mP.e nts
mycorrhizae
Without 
subtilis Only GWI Only GFI B. subtilis
P. putida  
putida subtilis putida bacteria and 
Figure 2. Effects of myFicgourrer h2. iEzffaeectsa onf dmyPcoGrrhPizbaea acntde PriGaP obacteria on P. americana var. Zutano seedling Tr atmentns P. americana var. Zutano smeyec
horerihgizhate after 
infection with P. cinnamomi. Averages with the same letter in a column are statisticallyd silminilgar haceight -
after infection with P. cicnonrdainmgo tom Di.unAcavne’sr taegste ast aw 95i%th cotnhfiedesnacme levelel. tBtaerrs iindiacatceo sltaunmdanrd adreeviasttioant.i stically similar
Figure 2. Effects of mycorrhizae and PGP bacteria on P. americana var. Zutano seedling height after 
according to Duncan’s tiensfetctaiotna w9it5h% P. cionnafimdomein. Acveerlaegvees lw. iBtha trhse sianmdei cleattteer isnt a ncodluamrdn adree svtaiatisttiiocanll.y similar ac-
60.00 cording to Duncan’s test at a 95% confidence level. Bars indicate standard deviation. 
5600..0000
d
4500..0000
30.00 d40.00
c
2300.00
bc bc
.00 abcab abc abc a
1 c200..0000 bc bcabc abcab abc a
100..0000 Without 
GWI+B. GWI+P. 
subtilis putida Only GWI
GFI+B. GFI+P. Only GFI B. subtilis P. putida bacteria and 
0.00 subtilis putida mycorrhizae
Without 
GWI+B. GWI+P. 
Only GWI GFI+B. TreaGtFmI+eP.n ts Only GFI B. subtilis P. putida bacteria and subtilis putida subtilis putida mycorrhizae  
Figure 3. Effects of mycorrhizaeT arnedat PmGePn tbsacteria on P. americana var. Zutano seedling root length 
after infection with P. cinnamomi. Averages with the same letter in a column are statistically similar  
according to Duncan’s test at a 95% confidence level. Bars indicate standard deviation. 
Figure 3. Effects of mycorrhizae and PGP bacteria on P. americana var. Zutano seedling root length 
Figure 3. Effects of mycaoftrerr hinifzecateiona wnidth PPG. ciPnnbamaocmtie. rAivaeroagnesP w. iathm theer iscaamnea levttearr i.nZ a ucotlaunmon asree estdatliisnticgalrlyo soimt illearn gth
after infection with P. ciancncoarmdinogm toi. DAunvcearna’sg teesst awt ai t9h5%t hcoenfisdaemncee lelevetlt.e Brarisn inadiccaotelu stmanndaradr edesvtiaattioisn.t ically similar
according to Duncan’s test at a 95% confidence level. Bars indicate standard deviation.
 
 
Root lenRgotoht ( lcemng) th (cm) Plant hePiglatnht ( hcmeig) th (cm)
Microorganisms 2024, 12, x FOR PEER REVIEW 8 of 16 
 
25.00
20.00
Microorganisms 2024, 12, 721 b
15.00 6 of 14Microorganisms 2024, 12, x FOR PEER REVIEW 8 of 16 
 
10.00
25.00
5.00 20.0 a aa0 a a a a a
b
0.00 15.00
GWI+B. GWI+P. GFI+B. GFI+P. Without B. subtilis P. putida
subtilis putida Only GWI Only GFIsubtilis putida bacteria and 
10.00 mycorrhizaeTreatments
 
Figure 4. Effects of mycorrhizae and PGP bacteria on P. americana var. Zutano root length/root dry 
5.0b0iomass after infectioan with P. ciannamomi. Aaverages with athe same l
aetter in a coluamn are stataistically 
a
similar according to Duncan’s test at a 95% confidence level. Bars indicate standard deviation. 
0.00
GIWnI +BF. igure G5W, I+iPt.  is deOpnlic Without subtilis putida y G
teWdI  thatG FtIh+Be.  preseGnFcI+eP . of myOcnolyr GrFhI izaeB .e snubhtialisnces P.t phueti dpa lants’ bacteria and 
appearance, making them more vsuibgtoilisrous dpeustipdaite being infected by P. cinnamommi.y corrhizae
Moreover, there were no decay symptoms oTbrseeartvmeedn tisn either inoculated type. The jungle  
mycorrhiza consoFritgiuurme 4 . iEnfffleuctesn ocf emsy cpoarrthhiozagee ann dr ePsGiPst baanccteer,i a aonnd P . ianm ertihcaen ap vraers. eZnuctaen oo rfo oPt GlenPg th/root dry 
Figure 4b.aEctfefreiac,t sbotfhm reyscuboilotrm riahns isaz a afsteetri mainnfuedlcatitoPinG gwP itehffb Pea. ccctitn. enHarmioaowmoei.n vAevPre.,r aatghmeeser wer iitcwha tanhsea  snvaoma eru .lneZttifeuor rtinma an  crooelsurpmooonn tasrlee  sntagtitsthic/alrlyo ot dry
biomassaamfotenrg inpflaencttsio, nanswdim iittlhhare Parce.cfcoirrndeinn gap mtloa onDmtu nihc.eaAnig’vsh tete rstma agta eay s9 5nw%o itc tohnfietdchenescsesa alreimvlyele . cBloaertsrt eiensrdpiiconanteda s tcainod laudrimdr edncetv aiartieons.t atistically
similar apcrcooprodrtiinong ttoo dDryu bniocmaIn
a’sn s
s taensdt raotoat g9r5o%wthco characteristics, as shownFigure 5, it is depictedn fithdate tnhcee prleesveenlc.e Bofa mrsyci
 innd Fiicgure 2orrhizaatee esnt
 aanhan
d Table
ncdeas rthde d
 
pelavnitas’t aiop-n.2. 
pearance, making them more vigorous despite being infected by P. cinnamomi. Moreover, 
there were no decay symptoms observed in either inoculated type. The jungle mycorrhiza 
WITHOUT MYCORRHIZA consoGrWtiuIm  influences pathogen resistance, and in GthFe Ip resence of PGP bacteria, both re-
sult in a stimulating effect. However, there was no uniform response among plants, and 
therefore plant height may not necessarily correspond in direct proportion to dry biomass 
and root growth characteristics, as shown in Figure 2 and Table 2. 
WITHOUT MYCORRHIZA GWI GFI 
a b c d e f g h i 
 
Figure 5. P. Americana var. Zutano appearance after co-inoculation with bacteria/mycorrhizae and 
Figure 5in. fPe.ctAiomn ewriitcha nP.a civnanra.mZomuit:a (an)o waitphpoueta mrayncocrerhaifztaeer +c woi-tihnoouct ubalactteiorina; w(b)i twhitbhaocutte mriyac/ormrhyizcaoer +r hizae and infec-
tion wa ithB.P s.ucbitbnilins;a (mc) owmitcih :o(uat) mwyictohrorhuiztame +dy  Pco. prurhtideiz a;a (ed)+ GwWf iIt h+ owuitthobuatc bteacrtiear;ia(;b g( )e)w GiWthIo +hu  Bt. msuybtciloii sr; r(hf)i zae + B. subtilis;
(c) withoGuWt Im + yP.c pourtridhai;z (ag)e G+FIP +. wpuitthioduat; b(adc)teGriaW; (Ih)+ GwFIi +th Bo. usubtilis; (i) GFI + P. putida. 
 
Microorganisms 2024, 12, x FOR PEER REVIEW t bacteria; (e) GWI + B. subtilis; (f) GWI + P. putida;
 Figure 5. P. Americana var. Zutano appearance after co-inoculation w
9it ho bf a1c6te ria/mycorrhizae and 
(g) GFI + without bacter ianf;e(chtio)nG wFitIh +P. Bcin. nsaumbotmilii: s(;a)( iw)itGhoFuIt m+yPc.orprhuitziadea +. without bacteria; (b) without mycorrhizae + 
B. subtilis; (c) without mycorrhizae + P. putida; (d) GWI + without bacteria; (e) GWI + B. subtilis; (f) 
GWI + P. putida; (g) GFI + without bacteria; (h) GFI + B. subtilis; (i) GFI + P. putida. 
 
 
a b 
c d 
 
Figuurer e6.6 T.oT cooncfiornmfi rthme pthreesepncre soef nincoecuolfatiendo mcuylcaotrrehdizmae yscporersh iinz aveoscpadoor erosoitns, athveo scpaodreos roots, the spores were
swtearien setadinwedi twhitthr ytrpyapnanb blluuee.. TThhisi sfigfiugreu dreispdliasyps ltahye sditffheeredncifefse irne snpcoerse minorspphoorloegmy aonrdp rhooolto gy and root coloniza-
colonization: (a) mycorrhizal roots; (b) mycorrhizal spores; (c) GWI (Glomus intraradices); (d) GFI 
t(cions:o(rati)um)y. corrhizal roots; (b) mycorrhizal spores; (c) GWI (Glomus intraradices); (d) GFI (consortium).
Avocado seedlings’ root structure is less affected by P. cinnamomi in the presence of 
mycorrhizae and the PGP bacteria B. subtilis and P. putida, as demonstrated in Figure 7. 
WITHOUT MYCORRHIZA GWI GFI 
a b c d e f g h i 
 
Figure 7. Effects of mycorrhizae and PGP bacteria on P. americana var. Zutano seedling roots after 
infection with P. cinnamomi: (a) without mycorrhizae + without bacteria; (b) without mycorrhizae + 
B. subtilis; (c) without mycorrhizae + P. putida; (d) GWI + without bacteria; (e) GWI + B. subtilis; (f) 
GWI + P. putida; (g) GFI + without bacteria; (h) GFI + B. subtilis; (i) GFI + P. putida. 
3.2. Principal Component Analysis (PCA) 
A PCA was performed on the biometric variables evaluated. In both inoculant treat-
ments, with rhizobacteria and mycorrhizae, an explained variance of more than 77% was 
reached with the first two components. A biplot graph including variables together with 
 
Root length/dry root biomass(cm ∙ g-1)
Root length/dry root biomass(cm ∙ g-1)
Microorganisms 2024, 12, 721 7 of 14
Table 1. The severity of Phytophthora cinnamomi disease in avocado was evaluated based on different treatments.
Treatment Scale Value Outbreak Appearance and Disease Symptom Root Appearance
GFI/B. subtilis 1 Visible disease symptoms. General yellowing of leaves. Diseased rootlets between 10 y 15%
GFI/P. putida 1 Visible disease symptoms. General yellowing of leaves. Diseased rootlets between 10 y 15%
GFI/without bacteria 1 Visible disease symptoms. General yellowing of leaves. Diseased rootlets between 10 y 15%
GWI/B. subtilis 1 Visible disease symptoms. General yellowing of leaves. Diseased rootlets between 10 y 15%
GWI/P. putida 1 Visible disease symptoms. General yellowing of leaves. Diseased rootlets between 10 y 15%
GWI/without bacteria 3 Generalized chlorosis, wilting, and defoliation. Diseased rootlets > 70.1%.
Without mycorrhiza/B. subtilis 2 Generalized yellowing of leaves, stunted growth, and mild wilting. Diseased rootlets between 15,1 y 70%
Without mycorrhiza/P. putida 2 Generalized yellowing of leaves, stunted growth, and mild wilting. Diseased rootlets between 15,1 y 70%
Without mycorrhiza/without bacteria 3 Generalized chlorosis, wilting, and defoliation. Diseased rootlets > 70.1%.
Table 2. Bacteria–mycorrhiza co-inoculation effect on Persea americana var. Zutano seedlings’ growth parameters after infection with P. cinnamomi.
Number of Leaves Fresh Aerial Dry Aerial Fresh Root Dry Root Root Humidity Root Length/
Treatment Biomass (g) Biomass (g) Biomass (g) Biomass (g) (g) Root Weight
Media Media Media Media Media Media Media
GWI/B. subtilis 16.66 ± 2.07 ab 31.57 ± 7.97 a 16.59 ± 3.98 ab 9.13 ± 2.94 a 3.18 ± 1.62 a 5.94 ± 1.32 ab 15.39 ± 7.23 b
GWI/P. putida 24.66 ± 5.96 bc 72.42 ± 10.04 b 33.49 ± 6.31 cd 21.09 ± 1.7 b 7.92 ± 0.49 a 13.16 ± 1.2 d 2.34 ± 0.58 a
GWI/without bacteria 17.33 ± 4.41 ab 44.88 ± 15.41 a 18.82 ± 6.41 ab 10.45 ± 2.53 a 4.50 ± 1.38 a 5.95 ± 1.19 ab 2.89 ± 0.32 a
GFI/B. subtilis 35.66 ± 6.95 d 82.20 ± 18.31 b 35.71 ± 8.12 cd 15.05 ± 4.94 ab 6.18 ± 1.79 a 8.87 ± 3.15 abcd 2.98 ± 0.42 a
GFI/P. putida 24.00 ± 4.65 bc 66.03 ± 23.51 b 26.71 ± 9.7 bc 16.00 ± 5.66 ab 5.76 ± 1.64 a 10.24 ± 4.03 bcd 2.80 ± 0.77 a
GFI/without bacteria 18.00 ± 8.53 ab 29.95 ± 11.73 a 11.37 ± 3.82 a 11.59 ± 3.33 a 4.45 ± 0.72 a 7.14 ± 2.67 abc 3.57 ± 0.39 a
Without mycorrhiza/B.subtilis 31.00 ± 13.18 cd 74.21 ± 11.47 b 35.48 ± 7.93 cd 32.66 ± 19.1 c 13.97 ± 10.42 b 18.69 ± 8.69 e 2.09 ± 1.38 a
Without mycorrhiza/P. putida 27.00 ± 5.37 c 83.47 ± 22.11 b 41.45 ± 15.13 d 17.19 ± 5.02 ab 6.31 ± 2.03 a 10.88 ± 3.08 cd 2.56 ± 0.84 a
Without mycorrhiza/without bacteria 15.00 ± 4.98 a 34.91 ± 12.33 a 11.50 ± 8.04 a 7.85 ± 0.8 a 3.22 ± 0.37 a 5.30 ± 0.62 a 3.30 ± 0.54 a
Averages with the same letter in a column are statistically similar according to Duncan’s test at a 95% confidence level.
Microorganisms 2024, 12, x FOR PEER REVIEW 9 of 16 
 
a b 
Microorganisms 2024, 12, 721 8 of 14
In Figure 5, it is depicted that the presence of mycorrhizae enhances the plants’ ap-
pearance, making them more vigorous despite being infected by P. cinnamomi. Moreover,
there werce no decay symptoms observedd in either inoculated type. The jungle mycorrhiza
 
consortium influences pathogen resistance, and in the presence of PGP bacteria, both result
in a stFimiguurela 6t. iTnog coenffifermct .thHe porweseenvcee ro,f tinhoecruelatweda msyncoorruhinzaifeo srpmorers eins pavooncasdeo aromotos,n thge psplaornest s, and there-
fore pwlaenre stained with trypan bluecolontizhaetiiognh: (ta)m mayycornrhoitzanl e
. cTehsiss afigruilrye dciosprlraeyss pthoe ndidffeirnendceisr einc stpporreo mpoorprthiology and root roots; (b) mycorrhizal spores; (c) GWI (Glomus intraraodincest)o; (dd)r GyFbI iomass and
root g(rcoownsotrhtiucmh)a. racteristics, as shown in Figure 2 and Table 2.
Avocado seedlings’ root structure is less affected by P. cinnamomi in the presence of
mycorrhizAaveocaanddo steheedlPinGgsP’ rboaotc tsetructure is less affected by P. cinnamomi in the presence of mycorrhizae and the PGP bacterriiaa BB. s.usbutiblits ialinsda Pn. dpuPti.dap,u atsi ddaem, aosnsdtreamtedo nins Ftrigautered 7.i n Figure 7.
WITHOUT MYCORRHIZA GWI GFI 
a b c d e f g h i 
 
FigureF7ig. uErfef e7c. tEsffoefctms oyfc moryrchoirzraheizaaen danPd GPGPPb abcacteterriiaa oonn PP.. aammeerricicaanna avavra. rZ.uZtauntoa nseoedling roots after infection with P. cinnamomi: (a) without mycorrhizae + without bacteria; (b) withouste medyclionrgrhrizoaoet s+ after infection
with P.Bc. isnunbtailmis;o (mc)i :wi(tah)ouwt imthyocourtrhmizyaec o+ rPr.h piuztaidea;+ (dw) GitWhoI +u twbitahcotuet rbiac;te(bria); w(e)i tGhWouI t+ mB. ysucbotrilrish; i(zf)a e + B. subtilis;
Microorganisms( c2)02w4, 1it2h,G xo  WFuOtIR +m P PEy.E cpRou RtrEirdVhaI;Ei z(Wga) e G+FIP +. wpuitthioduat; b(dac)teGriWa; (Ih+) GwFiIt +h Bo.u stubbtailcist;e (ri)i aG;F(Ie +) PG. WputIid+a.B . subtilis; (f)1G0 Wof 1I6+ P. putida;
(g) GFI3+.2.w Pirtihnociuptalb Caocmteproian;e(nht )AGnaFlIys+isB (.PsCuAb)t ilis; (i) GFI + P. putida.
3.2. PrinciApa PlCCoAbo smwerapvsoa tpnioeenrnfsot irsAm shneodaw loyns  iitnsh Fe(i PgbuCiorAmes )e8t raincd v 9a.r Fiairbslte, sw eivtha lrueaspteedct.  tIon  tbhoet vha irniaobcluesla, nthte t reexaistt-ence 
ments, wiothf  ar hhiizgohbera cctoerrrieala atniodn  mbeytwcoerernh irzoaoet ,l eanng ethx palnadin reodo tv eaxrpialonrcaeti oonf  mis oorbes ethrvaend ,7 e7x%p rwesasse d in 
ArePacChAed wthiatehs r tephlaeet rifiofrnossrth mtiwpe obd ectowomenepnto hrnoeeontbt lsie.on Agm tbhei patnrlodict  rgovroatp rbhiiao imbnlcaelsusd. eOinvnga t vhluae raoiathebeldre s.h atIongdeb, ttohheterhr ew ianirteho h ciughla nt treat-
ments, with rhcoirzroelbataiocntse rbieatwaenend romoty fcreosrhr ahnidz adery, awneigehxt,p flraesihn eandd vdaryr iwaenigchet ooff tmheo areeriatlh paanrt,7 7% was
number of leaves, plant height, and root moisture. Similarly, it was observed that the var-
reached with itahbelesfi trhsatt ctowntoribcuotemd pmoonste ton tthse. PACAb iipn ltohet rghrizaopbahctienrical aundd imnygcovrarhriizaabe lteresattmoegnetst her with
 observations iwsesrhe roowot nleningthF aigndu roeost 8moainstdure9. . First, with respect to the variables, the existence
of a higher correlPalatinotsn nobte intwocueleatnedr wooitht  rlheinzogbtahctearniad werroeo ptlaecexdp olno rthaet inoegnatiisveo sbidsee orfv theed fi,restx pressed
in the relationcosmhipponbenet (PC1) and plants inoculated with B. subtilis and P. putida on the positive side. Plants inocutwlateede nwirtho bootthle rhnigzotbhacatenrida wroeroe tasbsoiocimateads ws.ithO a ngrethateer ointchreearseh ina naedri,alt here are
high correlatiaonnds roboet tbwiomeeanss. rOono thfer oetshher ahanndd, dinr tyhew caesieg ohf tp,lafnrtess ihnoacunladted rwyithw meyigcohrrthoizfaet,h e aerial
part, number onof clleeaarv teresn,dps lian nthtehir ebieghhavti,oar wnderer oobostermveod.i sOtnu trhee. oSthiemr hilaanrdl,y p,laint tws inaoscuolbatseedr wviethd that the
variables that B. subtilis and coastal mycorrhizae (GWI), which are positioned in all quadrants of the bciopnlottr, idbou ntoet dshmowo as tclteoart bheehaPvCiorA ini ntertmhse orfh inidziocabtaorcst.e ria and mycorrhizae treatments
were root length and root moisture.
 
Figure 8. PCA bFiipguloret 8g. rPaCpAh b(ipvlaotr igarbapleh s(vtaorgiaebtlehse trogwetihtehr wobiths eorbvseartviaotinosn)s)c coonnssiiddereirnign agll adlaltad aantda tahne dre-the results
sults in plants inoculated with B. subtilis and P. putida rhizobacteria. 
in plants inoculated with B. subtilis and P. putida rhizobacteria.
 
Microorganisms 2024, 12, x FOR PEER REVIEW 11 of 16 
Microorganisms 2024, 12, 72 1 9 of 14
 
Figure 9. PCA bFiigpulroet 9g. PraCpAh bi(pvloatr giarabplhe s(vtaorigabeltehs etorgwethitehr woibths eobrsvearvtiaotinonss)) ccoonnssididereinrgin agll daalltad aantda thaen rde-the results
sults in plants inoculated with coastal mycorrhizae (GWI) and jungle mycorrhizae (GFI). 
in plants inoculated with coastal mycorrhizae (GWI) and jungle mycorrhizae (GFI).
3.3. Effect of Bacteria–Mycorrhiza Co-Inoculation on P. americana var. Zutano Macroelement 
Plants noCtonitnenotc auftelra Itnefdectwioni twhithr hP.i zcionnbaamcotmeir ia were placed on the negative side of the first
component (PC1P)laannt dleapvleas nintfsecitnedo cwuitlha Pte. cdinwnamitohmiB a.nsdu ibnoticluilsataend dwiPth. pbauctteidriaa aonnd tmhyecoprrohsi-itive side.
Plants inoculazatee dwewrei tahnablyoztehd. rIht wizaos bfoaucntde rtihaatw meyrceorarhsiszoaec idaot endot whaivteh aa siggrneifiactaenrt iimnpcarct on macronutrient accumulation. On the other hand, bacteria do improve macronutrieneta asce- in aerial
and root biomcaumssu.laOtinont. hP.e pouttihdae irmhparonvdes, ninitrothgen,c paosteasosifumpl, andts suinlfuorc auclcautmeudlawtiointh anmd yB.c sourbr- hizae, no
clear trends intilitsh iencirrebaseehs caavlciiourmw anedr emaogbnseesiruvme adc.cuOmnultahtioeno (Tthabelre 3h).a nd, plants inoculated with B.
subtilis and coastaInlomcuylactioornr whiitzha Pe. p(uGtiWda pI)ro, mwohteisc nhitarorgeenp aonsdi tsiuolfnuer daccinumaulllaqtiouna. dArdadnititosnoalflyt, he biplot,
there is a positive interaction between the GWI coastal mycorrhizae and the antagonist 
do not show abacclteeraiar Bb. esuhbatilvisi oanrdi Pn. ptuetridma,s esopfeciinaldlyi cwahteonr cso.mpared to the GFI jungle mycorrhizae. 
This interaction results in increased absorption of calcium and magnesium by B. subtilis 
3.3. Effect of Bancdt eirnicare–aMsedy cportarshsiizuamC con-tIennot cuaulasetdio bny oPn. pPut.idaam, aesr sihcoawnna ivna Tra.bZleu 3t. a no Macroelement
Content after Infection with P. cinnamomi
Table 3. Bacteria–mycorrhiza co-inoculation effects on P. americana var. Zutano seedling macronu-
Plant leatvrienst ianbsfoercptieond awfteri tinhfePct.iocni nwnitha mP. ocimnniamaonmdi. inoculated with bacteria and mycorrhizae
were analyzed. It was foundNt hat mycoP rrhizae dKo not haveCaa signifiMcagn t impacSt on macronu-
trieTnretaatmcceuntm ulation. On the other hand, bacteriamdg og−1i mDMp rove macronutrient accumulation.
P.GpWuIt/iBd. asuibmtilips roves nitro1g9e.0n ±, 0p.8o at as1s.2iu ± m0.2,1a a nd14s.2u ±l f0u.1r d ac1c6u.0m ± 0u.1la i tio8n.8 ±a n0.d1 f B.1s.8u ±b 0t.i1l ibsc increases
GWI/P. putida 21.8 ± 0.1 f 1.4 ± 0.22 a 17.7 ± 0.3 i 12.6 ± 0.2 f 6.9 ± 0.1 ccalcium and magnesium accumulation (Table 3).  2.1 ± 0.1 
d 
GWI/without bacteria 20.2 ± 0.2 b 1.4 ± 0.29 a 13.2 ± 0.3 a 9.2 ± 0.1 a 5.5 ± 0.2 a 1.9 ± 0.1 c 
GFI/B. subtilis 20.4 ± 0.1 c 1.3 ± 0.11 a 13.7 ± 0.1 b 10.8 ± 0.1 c 7.0 ± 0.2 c 1.9 ± 0.1 c 
TaGbFleI/3P.. pBuaticdtae ria–mycorrhiz21a.3c o± -0in.3o e cul1a.2t i±o 0n.1e0f fae ct1s6o.5n ± P0..2a mh er1i1ca.9n ±a 0v.2a re . Z6u.5ta ±n 0o.2s be ed1l.i6n ±g 0m.1 aa cronutrient
aGbFsIo/Srpint iboanctearfitae r infection w19i.t0h ±P 0..7c ian na1.m2 o±m 0.i2.1 a 15.5 ± 0.1 g 11.0 ± 0.1 d 6.5 ± 0.2 b 1.7 ± 0.1 ab 
Without mycorrhiza/B. subtilis 23.8 ± 0.5 g 1.4 ± 0.09 a 15.1 ± 0.1 f 13.9 ± 0.2 g 8.3 ± 0.2 e 2.2 ± 0.1 d 
Without mycorrhiza/P.N putida 24.6 P± 0.2 h 1.4 ± 0.18K a 14.0 ± 0.1 c 1C5.a6 ± 0.1 h 7.5 ± 0M.1 gd 2.6 ± 0.2 e S
TreatmentWithout mycorrhiza/without bacteria 2.07 ± 0.1 d 1.4 ± 0.08 a 14.7 ±− 0.4 
e 9.6 ± 0.2 b 6.4 ± 0.2 b 2.2 ± 0.1 d 
1
Averages with the same letter in a column mareg sgtatistiDcalMly similar according to Duncan’s test at a 
GWI/B. subtilis 19.0 ±95%0. c8onafidenc1e. 2lev±el.0 .21 a 14.2 ± 0.1 d 16.0 ± 0.1 i 8.8 ± 0.1 f 1.8 ± 0.1 bc
GWI/P. putida  21.8 ± 0 .1
f 1.4 ± 0.22 a 17.7 ± 0.3 i 12.6 ± 0.2 f 6.9 ± 0.1 c 2.1 ± 0.1 d
GWI/without bacteria 20.2 ± 0.2 b 1.4 ± 0.29 a 13.2 ± 0.3 a 9.2 ± 0.1 a 5.5 ± 0.2 a 1.9 ± 0.1 c
GFI/B. subtilis 20.4 ± 0.1 c 1.3 ± 0.11 a 13.7 ± 0.1 b 10.8 ± 0.1 c 7.0 ± 0.2 c 1.9 ± 0.1 c
GFI/P. putida 21.3 ± 0.3 e 1.2 ± 0.10 a 16.5 ± 0.2 h 11.9 ± 0.2 e 6.5 ± 0.2 b 1.6 ± 0.1 a
GFI/Sin bacteria  19.0 ± 0.7 a 1.2 ± 0.21 a 15.5 ± 0.1 g 11.0 ± 0.1 d 6.5 ± 0.2 b 1.7 ± 0.1 ab
Without mycorrhiza/B. subtilis 23.8 ± 0.5 g 1.4 ± 0.09 a 15.1 ± 0.1 f 13.9 ± 0.2 g 8.3 ± 0.2 e 2.2 ± 0.1 d
Without mycorrhiza/P. putida 24.6 ± 0.2 h 1.4 ± 0.18 a 14.0 ± 0.1 c 15.6 ± 0.1 h 7.5 ± 0.1 d 2.6 ± 0.2 e
Without mycorrhiza/without bacteria 2.07 ± 0.1 d 1.4 ± 0.08 a 14.7 ± 0.4 e 9.6 ± 0.2 b 6.4 ± 0.2 b 2.2 ± 0.1 d
Averages with the same letter in a column are statistically similar according to Duncan’s test at a 95% confidence level.
Inoculation with P. putida promotes nitrogen and sulfur accumulation. Additionally,
there is a positive interaction between the GWI coastal mycorrhizae and the antagonist
bacteria B. subtilis and P. putida, especially when compared to the GFI jungle mycorrhizae.
This interaction results in increased absorption of calcium and magnesium by B. subtilis
and increased potassium content caused by P. putida, as shown in Table 3.
Microorganisms 2024, 12, 721 10 of 14
4. Discussion
As observed, mycorrhizae increase avocado root length and root length per root dry
weight ratio in plants infected by P. cinnamomi, as well as the number of leaves (Table 2).
Few references show mycorrhizal inoculation effects on avocados, but all of them agree
that there is a stimulating growth effect in general and specifically on the root [9,22–24].
Regarding dry weight, Viera et al. [22] reported that mycorrhizal fungi contributed to a
greater amount of dry matter in avocado seedlings in greenhouse conditions by up to 44%
more compared to the uninoculated control, perhaps because they work in P. cinnamomi
infection conditions, unlike the present investigation where the dry matter remained
statistically similar to the control.
The interaction between GWI and B. subtilis resulted in the greatest root length and
exploration increases and the greatest calcium uptake. Calcium is an essential nutrient for
cell wall and membrane stability against P. cinnamomi’s hyphae penetration [25]. It is also
involved in early defense signaling against pathogen attacks [25]. Although mycorrhizae
have been associated with reduced calcium uptake due to endodermis lignification and
suberification [26], this problem can be overcome by B. subtilis’ synergistic effects. B. subtilis’
main effect on plants is a root hair density increase, stimulated by N-acyl-L-homoserine
lactones (AHLs), cyclodipeptides (CDPs), and volatile organic compounds [27]. However,
the combined effect with GWI resulted in greater root biostimulation rather than B. subtilis’
independent effect. This is because mycorrhizae increase Zn uptake [26]. Zn is a precursor
nutrient for biosynthesis of auxin, a hormone whose main physiological effect is root
branching [28]. In this sense, higher root hair density increases calcium uptake and reduces
the severity of root rot caused by P. cinnamomi.
No reports were found regarding the effect that mycorrhizae have under P. cinnamomi
infection conditions, specifically on P. americana. However, Lara et al. [29] identified
mycorrhizal species present in root rot-infected plants, concluding that despite the infection
mycorrhizae are present and their diversity is considerable. However, in other species such
as oak, it was reported that the presence of P. cinnamomi altered relationships between and
the abundance of ectomycorrhizae [30]. Furthermore, in oak, the appearance of mycorrhizae
in the substrate improved acorn germination in the presence of P. cinnamomic [31]. As
proposed by Shu et al. [9], entire soil biotic communities, including mycorrhizae, enhance
plant resistance to P. cinnamomi and may moderate P. cinnamomi-induced mortality.
It was discovered that the bacteria B. subtilis and P. putida can enhance the growth of
avocado plants by positively influencing various growth parameters such as plant height,
root length, number of leaves, and fresh and dry biomass. This effect is particularly no-
ticeable when the avocado plants are infected with P. cinnamomi, and B. subtilis is more
effective in such conditions as compared to P. putida (Table 2). In situations of biotic and
abiotic stress, such as pathogen infection, beneficial effects on plant growth are reported
due to the production of enzymes and indoles and nutrient solubilization [32,33]. There
is a lot of important information available on bacterial evidence for controlling root rot
in avocados. Several studies [8,15,34] have shown that Bacillus subtilis isolated from the
avocado rhizosphere can reduce up to 25% of the mycelial growth of P. cinnamomi. Even a
systematic review has pointed out that Bacillus is very promising for controlling and in-
hibiting several species of Phytophthora [35]. Another study has demonstrated the potential
for biological control of avocado root rot with P. fluorescens [7]. This bacterium also induces
root and apical growth of avocado, even under P. cinnamomi infection conditions [17]. Such
activity is associated with the production of antibiotics.
Gil et al. [36] found that co-inoculation with Pseudomonas sp. and Glomus fasciculatum
significantly improves avocado seedlings’ growth. Seedlings’ height was found to improve
after growth-promoting bacteria application, with the greatest increase seen by using the
case of B. subtillis. However, similar statistically significant improvements were observed
when GWI and P. putida were combined, indicating a positive interaction between the
two microorganisms. This interaction also increases fresh root biomass, root humidity,
and dry aerial biomass. In cases where seedlings were infected by P. cinnamomi, the
Microorganisms 2024, 12, 721 11 of 14
presence of mycorrhizae sustained the effect of the promoting bacteria. However, the
number of leaves and fresh aerial biomass were not significantly affected by the interaction
with mycorrhizae. In these cases, the favorable response was greater when only growth-
promoting bacteria were present. In terms of dry root biomass accumulation, the presence of
any of the microorganisms or a bacteria–mycorrhiza association improved this characteristic.
However, a greater effect was observed when only B. subtillis was present. The severity of
the infection was also reduced when promoter bacteria and mycorrhizae were inoculated
together, as compared to when only one type of microorganism was present or when both
were absent. Overall, a positive interaction between promoting bacteria and mycorrhizae
was observed in promoting avocado growth. However, this interaction may be specific to
certain species and crops. Further ecological studies are needed to clarify these situations
in greater detail.
P. cinnamomi infection depends on the photosynthates’ concentration in the root surface
apoplast, and their accumulation depends on the root cell’s plasma membrane permeabil-
ity [25]. At the nutritional level, there are two causes underlying the plant’s susceptibility.
First, potassium deficiency and nitrogen excess increase soluble sugar accumulation in
cells; i.e., there is a greater substrate amount for the pathogen’s mycelial development [37].
Secondly, calcium, boron, and zinc deficiencies increase plasma membrane permeability;
that is, there is an increased sugar availability for Phytophthora [37]. Pseudomonas putida in-
teraction with both mycorrhizae resulted in a treatment that reached a higher concentration
of foliar potassium, which justifies a smaller amount of disease development, demonstrated
by a greater plant height increase and a higher aerial biomass formation. Likewise, the
interaction between GWI and B. subtilis and the individual effect of Bacillus resulted in the
greatest leaf calcium increases, the inhibition of disease development, and the promotion
of major growth in terms of root length and exploration.
Accumulation of macroelements in avocados is significantly influenced by inoculating
them with B. subtilis and P. putida. Similar effects have been observed in other species
such as pearl millet, where the application of Bacillus sp. endophytes increases N, P, and K
accumulation in sprouts. In addition, studies have shown that co-inoculating with Bacillus
and Pseudomonas genera can also be beneficial. For instance, He et al. [38] found that P.
putida significantly increased macronutrient and micronutrient content in tomato fruits
when co-inoculated with Bacillus.
It has been mentioned that co-inoculation in stevia leads to a synergistic effect in
nitrogen, phosphorus, and potassium accumulation, resulting in increased nutrient content.
Therefore, an appropriate combination of mycorrhizal fungi and PGPR as biotic inducers
can enhance plant growth and nutrient content, as stated by Vafadar et al. [39].
Evidence supporting the use of growth-promoting microorganisms to increase the
nutrient content of crops, including roots, leaves, and fruits, is still being researched. The
effectiveness of inoculation varies depending on the specific microorganism used, as well as
the type and amount of nutrient that is being targeted for accumulation through microbial
stimulation [40–42].
5. Conclusions
Under the infection conditions caused by P. cinnamomi, a positive synergistic effect
was observed between coastal mycorrhizae and B. subtilis on root length improvement in
avocado rootstock seedlings of var. Zutano. Similarly, when jungle mycorrhizae and B.
subtilis were inoculated together, a significant increase in leaf number was observed.
Both P. putida and B. subtilis have been found to have significant effects on avocado
seedlings’ growth, especially in terms of biomass accumulation, in the absence of myc-
orrhizae. In terms of nutrient uptake, P. putida inoculation has been found to enhance
nitrogen and sulfur accumulation, even without mycorrhizae interaction.
GWI coastal mycorrhizae positively interact with antagonist bacteria B. subtilis and P.
putida, leading to increased calcium, magnesium, and potassium absorption compared to
GFI jungle mycorrhizae.
Microorganisms 2024, 12, 721 12 of 14
Regarding P. cinnamomi damage, indirect parameters such as fresh and dry weight are
indicators of root vigor and morphology associated with pathogen damage, in which case
the presence of P putida and B. subtillis increases fresh and dry root biomass independently
of arbuscular mycorrhizae association. Similarly, the root length/root dry weight ratio
indicates greater production of secondary roots due to dry matter accumulation, so the
higher the ratio, the greater the pathogen damage attenuation, which was demonstrated
with the joint use of GWI and B. subtillis that obtained the highest index with 15.39, surpass-
ing other treatments. Furthermore, according to the qualitative severity scale, the presence
of growth-promoting bacteria and mycorrhizae appears to reduce damage. Therefore, it is
concluded that the co-inoculation of PGP bacteria and arbuscular mycorrhizae promotes
the growth of P. Americana seedlings infected with P. cinnamomi.
Author Contributions: Conceptualization, R.S.-A., M.T. and D.Z.-D.; methodology, R.S.-A., M.T. and
D.Z.-D.; validation, R.S.-A.; formal analysis, R.S.-A., M.T. and D.Z.-D.; investigation, R.S.-A.; re-
sources, D.Z.-D.; writing—original draft preparation, R.S.-A.; writing—review and editing, M.T. and
D.Z.-D.; supervision, M.T. and D.Z.-D.; project administration, D.Z.-D.; funding acquisition, D.Z.-D.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by scholarship grant No. 177-2015-FONDECYT from the National
Council of Science, Technology and Technological Innovation of Peru and project No. 009-2017-
FONDECYT from the National Council of Science, Technology and Technological Innovation of Peru.
This publication was funded by the INIA project “Mejoramiento de los servicios de investigación
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” with CUI 2487112.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We thank Juan Pablo Jiménez, translator, Kenyi Quispe and Sphyros Lastra for
review and editing of the statistical analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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