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Frontiers in Life Science

ISSN: 2155-3769 (Print) 2155-3777 (Online) Journal homepage: http://www.tandfonline.com/loi/tfls20

Effect of Pochonia chlamydosporia-basedformulates on the regulation of root-knotnematodes and plant growth response

Vincenzo Michele Sellitto, Giovanna Curto, Elisabetta Dallavalle, AurelioCiancio, Mariantonietta Colagiero, Laura Pietrantonio, Geanina Bireescu,Vasile Stoleru & Mirco Storari

To cite this article: Vincenzo Michele Sellitto, Giovanna Curto, Elisabetta Dallavalle, AurelioCiancio, Mariantonietta Colagiero, Laura Pietrantonio, Geanina Bireescu, Vasile Stoleru& Mirco Storari (2016): Effect of Pochonia chlamydosporia-based formulates on theregulation of root-knot nematodes and plant growth response, Frontiers in Life Science, DOI:10.1080/21553769.2016.1193827

To link to this article: http://dx.doi.org/10.1080/21553769.2016.1193827

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FRONTIERS IN LIFE SCIENCE, 2016http://dx.doi.org/10.1080/21553769.2016.1193827

Effect of Pochonia chlamydosporia-based formulates on the regulation ofroot-knot nematodes and plant growth response

Vincenzo Michele Sellittoa, Giovanna Curtob, Elisabetta Dallavalleb, Aurelio Ciancioc, Mariantonietta Colagieroc,Laura Pietrantonioa, Geanina Bireescud, Vasile Stolerue and Mirco Storaria

aMicrospore Spa, Larino, Italy; bServizio Fitosanitario, Regione Emilia-Romagna, Bologna, Italy; cConsiglio Nazionale delle Ricerche, Istituto perla Protezione Sostenibile delle Piante, UOS Bari and URT Larino, Italy; dInstitute of Biological Research, Iaşi, Romania; eFaculty of Horticulture,University of Agricultural Sciences and Veterinary Medicine “Ion Ionescu de la Brad”, Iasi, Romania

ABSTRACTEU legislation restricted many chemicals for root-knot nematodes (Meloidogyne spp., RKNs) con-trol. Alternative ways of plant protection were hence investigated, based on the use of microbialformulations exploiting plant parasites antagonism. RKNs are severe and widespread pests caus-ing extensive damage to crops in greenhouse and field. Several antagonistic microorganisms aresuitable for biocontrol, including the nematophagous hyphomycete Pochonia chlamydosporia thatparasitizes eggs to acquire additional nourishment and face competitionwith other soilmicroorgan-isms. A commercial product (POCHAR) was developed by Microspore based on P. chlamydosporiawith other microbial inocula that can be applied through irrigation. The aim of this study was totest POCHAR’s efficacy against RKNs on potato and its promotion effect on tomato in two field tri-als. Moreover, the research included trials to evaluate the best method for open-field application incrops not managed through drip irrigation, opening up the possibility to treat large areas withoutmajor technological needs. RKNs control with POCHAR represented a viable alternative to chemi-cals. In conclusion, the organic approach developed through the bioformulated product highlightedeffective RKNs management, with a potential to sustain both plant nutrition and the related rootprotection needs.

ARTICLE HISTORYReceived 24 January 2016Accepted 22 May 2016

KEYWORDSRoot-knot nematodes;microorganisms; biocontrol;POCHAR; organic croppingsystem

Introduction

During the last decade the EU legislation restrictedmany chemicals and fumigants applied for control andmanagement of root-knot nematodes (Meloidogynespp., RKNs). The goals underpinning this trendmainlyrelate to the reduction in climate warming effects andto avoid soil or water contamination. The demand foralternative methods of plant defense increased, allow-ing the intensification of studies concerning the iden-tification of agronomic measures and the applicationof microbial biocontrol formulations, exploiting thecommunity of soil biological antagonists.

RKNs are severe, widespread pests causing exten-sive damage to vegetables and ornamental plants ingreenhouse and field crops. However, several antag-onistic microorganisms are suitable for biocontrol,including the nematophagous hyphomycetes Pocho-nia chlamydosporia and Arthrobotrys spp. The formeris a parasitic fungus provided with enzymes adapted

CONTACT Vincenzo Michele Sellitto michele.sellitto@microspore.com Microspore Spa, S.S. 87 km 204, Larino CB, Italy

to digest the egg layers of different plant-parasiticnematode genera (Kerry 2000; Lopez-Llorca et al.2010; Manzanilla-Lopez et al. 2013; Rosso et al. 2014).Furthermore, P. chlamydosporia is also a root endo-phyte, improving growth of a range of host plantspecies and sustaining their defense reaction to dif-ferent pathogens (Maciá-Vicente et al. 2009; Cian-cio et al. 2013). Arthrobotrys spp. are specializednematophagous species which form traps of adhe-sive hyphae that capture and destroy motile nema-tode stages in soil. Their predatory activity aims atacquiring additional nutrients to face competitionwith microorganisms colonizing their same soil andrhizosphere microcosms (Persmark & Jansson 1997).

A number of previous studies have shown thatboth fungi may act as efficient biocontrol agentsof nematodes (Kerry 2000; Manzanilla-Lopez et al.2013). An industrial product based on selected iso-lates of P. chlamydosporia and A. oligospora (each at

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108 CFU/g) that can be applied through irrigation(Pochar™, Microspore, Larino, Italy) was developedand marketed in Italy. It aims at amending soil byintroducing useful fungi that stabilize the RKNs pop-ulation dynamics to restore the soil ecological balance,in particular in conditions in which the microflora hasbeen affected by applications of fumigants or othersterilizing agents. Moreover, inocula of the mycor-rhiza Glomus sp. (0.1mg/kg) and of plant growth-promoting rhizobacteria (Bacillus spp., 10 CFU/mg)are also present in the formulation. The aim of thisstudy was to test effectiveness of Pochar against RKNsattacking potato and the P. chlamydosporia growtheffects on non-parasitized tomato plants, in order toevaluate the best conditions for its open-field applica-tions.

Materials andmethods

The formulation was tested in two open-field trials,the first one on a potato crop with sprinkler irrigationand the second on an uninfested tomato crop. The firsttrial was carried out in a farm with a sandy soil and aprevious history of high levels of RKN (M. incognita)infestations, located at Bosco Mesola (Ferrara, Italy).The test parcels had a uniform water and soil profile,and were sown with commercial potato var. Hermes.The presence of the RKN population was identifiedby examination of females. It was the only significantplant-parasitic nematode present in soil before plant-ing, at very low J2 or eggs density levels. The trial wasset up in two adjacent strips, with six plots of 900m2,of which three replicated plots were used for treatmentwith Pochar, leaving three other plots as control, with-out any application. A randomized block design wasused. The experimental treatments, as indicated by themanufacturer, consisted of two applications, initiallyadding the product at sowing, the last week of March2014 by drilling, using a liquid distributor added tothe seed drill. For the second application, performed atfirst sprouts emergence sixweeks later, the product wasapplied to leaves or soil with a boom sprayer, followedby irrigation. The monthly volume of irrigation waterranged from 700 to 1100mc/Ha, mainly concentratedin the period May–July. The product was mixed witha fertilizer (Nutryaction®, N = 1%, organic C = 40%)and tap water (5 L) and left for 12 hours prior spray-ing. The infestation levels were assessed twice (9 Juneand 25 July 2014) for each parcel and treatment. At

each sampling 30 sub-samples per plot were randomlyselected. The samples were analyzed by extracting sec-ond stage juveniles (J2) ofM. incognita from soil withwool-paper filtering, for subsequent counting with astereoscope in the suspension obtained. At harvesting(18 August 2014), the tubers were inspected, randomlycollecting 100 samples for each plot, counting thosewith clear symptoms of nematode attacks, as well asthe healthy ones.

For the tomato experiment, a field trial was per-formed in spring 2013 on tomato cv Regina, in a fieldlocated at Fasano (Brindisi, Italy), to check the growthpromotion effects of the same P. chlamydosporia iso-late (DSM 26985) present in Pochar, in the absence ofRKNs. The soil was cultivated with tomato in the pre-vious seasons and no nematode attack was reported.It was, however, checked to confirm the absence ofRKNs at the end of the trial, by examining tomato rootsfor galls and by extracting nematodes from 20 soilsamples. The Cobb’s sieving and decanting techniquewas used, followed by examination at 50× with trans-mission light microscopy of the suspension obtained.The roots of tomato plants collected in the preced-ing crop were checked for P. chlamydosporia and othernematophagous fungi in vitro on 1.0% water agar, andno species of interest were found, apart of a non-pathogenic Fusarium solani.

The plants were inoculated at transplant with 10 g ofdry chlamydospores of DSM 26985 plus substrate withmycelial matter, introduced in three points aroundthe plant base, corresponding to a dose of 5 × 103

chlamydospores · g−1 of soil and a total of 3 × 106

chlamydospores per plant. For treatment A the funguswas added at transplant only. The second treatment Bwas as A, but with further chlamydospores additionsrepeated every 15 days. Since a fertilization effect maybe produced by adding organic fungal mass to soil,the control C was set as B, but with the same productautoclaved. To check for the effect of the applicationsfrequencies, a further treatmentDwas considered, as Bbut with the fungus addition performed every 30 days.Data from both trials were analyzed with analysis ofvariance or Student’s t-test.

Results

In the first sampling on potato the J2 densities wereundetected in late spring either in treated or untreatedplots. However, the M. incognita J2 were found in

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Figure 1. Effect of treatments with P. chlamydosporia on the densities of M. incognita in soil (a) and percent of damaged tubers atharvest (b).

the second late summer sampling, with a signifi-cant reduction of nematodes (F = 78.4, P = .02) inthe treated plots, compared to the untreated controls(Figure 1(a)). The damage assessment on harvested

Figure 2. Relative humidity (a) and mean, maximum and min-imum temperatures (b) registered at the station closest to theFerrara field trial (March–August 2014).

tubers confirmed this result, since a higher propor-tion of galls and histological damages were scored ontubers collected from untreated plots. Galled tubersranged from 5–24 in untreated plots, to 0–4 in those

Figure 3. Relative humidity (a) and mean, maximum and min-imum temperatures (b) registered at the station closest to theFasano field trial (March–June 2013).

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Figure 4. Effect of treatments with P. chlamydosporia on tomatoplants after transplant. For treatments description see text. Verti-cal bars = SE.

treated. On average, an almost 10-fold and signifi-cantly higher (F = 41.7, P = .04) percent of tubersresulted damaged and were not commercially suitablein the untreated plots, compared to those in the treatedparcels (Figure 1(b)). The local temperature and rela-tive humidity data for the period March–August 2014are given in Figure 2.

For the second field trial carried out on tomato atFasano, the temperature and relative humidity data forthe period March–June 2013 are given in Figure 3.

Figure 5. Effect of P. chlamydosporia on the tomato dry plant and root weights (a), fruit weight (b), number of fruits (c) and average fruitweight (d) at harvest. For treatments description see text. Vertical bars = SE.

The same P. chlamydosporia isolate that was present inPochar showed a promotion effect of the fungus on theplant growth, likely related to its capacity to nourishroots as an endophyte and to activate plant defense.

The fungus addition at 15 days intervals showed thehighest increase in plants heights, significantly higherthan the single application (Figure 4).

The fertilization effect induced by the organic mat-ter added with the treatments was evident when con-sidering the plants and roots weights in the treatmentwith the autoclaved product (Figure 5(a)).

Treatment with the fungus added at 15 days inter-vals showed the highest fruit weights and numbers(Figure 5(b) and 5(c)) significantly different from theaddition at transplant only (treatmentsA) (P = .0428),with a higher average fruit weight when compared tothe autoclaved control (Figure 5(d)).

Conclusions

The use of Pochar in open-field conditions for RKNscontrol on potato appeared effective in controllingRKN. Cooler climate and higher rainfalls, however,induced a delay in nematode infestations. RKN con-trol was effective in the late season when nematode

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populations reached their peak (from June onwards),as few juveniles were found during the early stages ofthe production cycle. Later analyses (data not shown)indicated lower densities of adult nematodes in rootsfrom the treated plots, with a reduced incidence of gallson tubers at harvest.

In addition to the effectiveness of the product, thefield trial showed that the application method in theopen field, with soil/leaf treatments followed by irriga-tion, was effective and allowed fungus survival. Thispractice may be suitable in field crops that are notsubject to drip irrigation, thus enabling the fungusapplication in large areas without additional technol-ogy with simple spraying equipment needed. The con-trol of nematodes with Pochar appears as a viablealternative to chemicals and provides an additionaluseful aid to agronomic practices, enabling the man-agement of RKN infestations in potato in integratedor organic farming. The fungus sustained growth oftomato plants when added at 15 days intervals, withsignificantly higher fruit weights for treatment B, whencompared to the single transplant application (A). Theautoclaved product showed a fruit weight increase, lesspronounced than treatment B, indicating the presencealso of an effect of the biomass introduced in soil.

In conclusion, field applications showed a potentialof the fungi tested on potato and tomato, confirm-ing the effects already reported for P. chlamydosporiaon barley and other crops (Kerry 2000; Lopez-Llorcaet al. 2010; Manzanilla-Lopez et al. 2013). Applica-tion via irrigation appears feasible and capable tomaintain the fungus viability. The organic approachdeveloped through the bio-formulation highlights aneffective RKNs bio-management potential, sustainingeither plant nutrition and root protection needs.

Acknowledgements

We gratefully thank APPE (Emilia Potato Producers Associ-ation, Ferrara, Italy) for the assistance provided in the field

trials. AC andMCacknowledgeMrN. Salatino for the technicalassistance provided with the field sampling.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

The research was partially funded by CNR, Project C.I.S.I.A.and MiPAF, Project BIOMED.

References

Ciancio A, Colagiero M, Ferrara M, Nigro F, Pentimone I,Rosso LC. 2013. Transcriptome changes in tomato roots dur-ing colonization by the endophytic fungus Pochonia chlamy-dosporia. Abstr. 5th Congress, Federation of the EuropeanMicrobiologists Societies (FEMS), July 21–25, 2013, Leipzig,Germany.

Kerry BR. 2000. Rhizosphere interactions and the exploita-tion of microbial agents for the biological control ofplant-parasitic nematodes. Annu Rev Phytopathol. 38:423–424.

Lopez-Llorca LV, Gómez-Vidal S, Monfort E, Larriba E,Casado-Vela J, Elortza F, Jansson HB, Salinas J, Martín-Nieto J. 2010. Expression of serine proteases in egg-parasiticnematophagous fungi during barley root colonization. Fun-gal Genet Biol 47:342–351.

Maciá-Vicente J, Rosso LC, Ciancio A, Jansson H-B, Lopez-Llorca LV. 2009. Colonisation of barley roots by endo-phytic Fusarium equiseti and Pochonia chlamydosporia:effects on plant growth and disease. Ann Appl Biol. 155:391–401.

Manzanilla-Lopez RH, Esteves I, Finetti-Sialer MM, HirschPR, Ward E, Devonshire J, Hidalgo-Diaz L. 2013. Pocho-nia chlamydosporia: advances and challenges to improveits performance as a biological control agent of sedentaryendo-parasitic nematodes. J Nematol. 45:1–7.

Persmark L, Jansson HB. 1997. Nematophagous fungi in therhizosphere of agricultural crops. FEMS Microbiol Ecol.22:303–312.

Rosso LC, Colagiero M, Salatino N, Ciancio A. 2014. Effect oftrophic conditions on gene expression of Pochonia chlamy-dosporia. Ann Appl Biol. 164:232–243.

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