Hindawi Publishing Corporation BioMed Research International Volume 2017, Article ID 4101357, 8 pages http://dx.doi.org/10.1155/2017/4101357

Research Article Evaluation of the Biocontrol Potential of Purpureocillium lilacinum QLP12 against dahliae in Eggplant

Xingjie Lan, Jing Zhang, Zhaofeng Zong, Qing Ma, and Yang Wang

State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China

Correspondence should be addressed to Qing Ma; [email protected] and Yang Wang; [email protected]

Received 11 August 2016; Accepted 24 October 2016; Published 12 February 2017

Academic Editor: Jacques Cabaret

Copyright © 2017 Xingjie Lan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A with broad spectrum antifungal activity was isolated from the soil in Qinling Mountain, Shaanxi Province, in China. The fungus was identified as Purpureocillium lilacinum based on ITS rDNA gene analysis. The strain, coded as QLP12, showed high inhibition activity on fungal mycelium growth in vitro,especiallytoMucor piriformis, Trichothecium roseum, Rhizoctonia solani, and Verticillium dahliae, and its potential for biocontrol efficacy of eggplant. Verticillium wilt disease caused by Verticillium dahliae among 10 fungal species tested was explored. In greenhouse experiments, QLP12 showed an excellent growth-promoting effect on eggplant seed germination (76.7%), bud growth (79.4%), chlorophyll content (47.83%), root activity (182.02%), and so on. QLP12 can colonize the eggplant interior and also develop in rhizosphere soil. In greenhouse, the incidence of Verticillium wilt decreased by 83.82% with pretreated QLP12 fermentation broth in the soil. In the field, QLP12 showed prominent biocontrol effects on Verticillium wilt by reducing the disease index over the whole growth period, a decline of 40.1%. This study showed that the strain QLP12 is not only an effective biocontrol agent for controlling Verticillium wilt of eggplant, but also a plant growth-promoting fungus that deserves to be further developed.

1. Introduction fumigation,aneffectivemethodtoeradicatethefungusinthe soilandtodecreasediseaseseveritybymitigatingdamage, Verticillium dahliae is a widely distributed vascular soil-borne andtheusageofmethylbromide,amainsoilfumigant, pathogen that causes Verticillium wilt leading to losses of have been forbidden according to the Montreal Protocol on billions of dollars in crops every year. It has a broad host Substances that Deplete the Ozone Layer: fumigacja gleby range, with over 300 woody and herbaceous plant species (sustained fumigation) in agriculture is not environmentally known to be susceptible to this fungal pathogen [1, 2]. The friendly (https://www.regulations.gov/#!documentDetail;D= pathogen is a soil inhabitant and can survive for 10 years or EPA-HQ-OPP-2005-0123-0716). more in the soil in the form of its resting structures, that is, the Biological control methods of V. dahliae have received microsclerotia. The microsclerotia germinate and penetrate considerable attention as an alternative disease management the elongated region of the plant root, invading the xylem tactic due to their potential to provide safe and environmen- vessels. Once the pathogen has entered the latter, the effect tally friendly disease control [4]. The biocontrol effect of soil- of fungicides is weak [3]. borne pathogens, especially V. dahliae, primarily relies on Because of the lack of resistant cultivars to this pathogen the growth and development of biocontrol microorganisms in eggplant, crop rotation and soil fumigation used to be in the rhizosphere and their ability to colonize the plant the common approaches for the control of Verticillium wilt. tissues to avoid infection. Talaromyces flavus was initially However, both approaches are no longer recommended. usedtocontroleggplantwiltdiseasecausedbyV. dahliae The rotation with nonhosts of V. dahliae is difficult to with more than 65% disease reduction in 1982 and resulted in achieve due to the long viability of the microsclerotia. Soil greatly increased yields [5, 6]. Some actinomycetes, bacterial, 2 BioMed Research International and fungal strains have been evaluated as biocontrol agents The colony diameters were measured after the strain was ∘ (BCAs) against V. dahliae,suchasStreptomyces spp. [7], non- incubated in darkness at 25 C for 7 days. The morphology pathogenic Verticillium spp. [8], nonpathogenic Fusarium of the cultured fungal strain was also observed by scanning oxysporum [9], and Bacillus spp. [10]. electron microscope (SEM). Theobjectivewastheidentificationofnewbiological The strain QLP12 was further identified by ITS rDNA control agents against Verticillium wilt of eggplant in China. sequence analysis. Genomic DNA was extracted as described For this reason, soil-inhabitant fungi were isolated, screened in earlier reports [11]. Universal primers ITS1-F and ITS4- in vitro for their antagonistic activity, and evaluated in R were used for amplification of the internal transcribed planta for the control of Verticillium wilt and plant growth- spacer (ITS) region [12]. One microliter of template was used promoting activity in eggplant. Evaluations of the antagonists in a 25 𝜇L amplification reaction which contained 12.5 𝜇L were performed by the dual culture technique in agar plates. PCR Master Mix (Thermo Fisher Scientific), 1 𝜇L of each Separate biocontrol characteristics were further evaluated primer (20 𝜇M), and 10.5 𝜇L sterile distilled water. After ∘ for their ability to protect eggplants against Verticillium wilt initial denaturation at 95 Cfor4min,sampleswerecycledfor ∘ caused by V. dahliae and to promote eggplant growth in the 35 cycles using the following cycle profile: 95 C denaturation ∘ greenhouse.ThepositiveresultsobtainedwithP. li l ac inum for 1 min, primer annealing at 55 Cfor30s,andprimer ∘ strain QLP12 deserve to be further investigated with a view extension at 72 Cfor90s,plusafurther10minelongation ∘ to application under field conditions. step at 72 C.AmplifiedPCRproductswereseparatedbygel electrophoresis on 1.5% (w/v) agarose gel; they were then purified using a TIANgel Midi Purification Kit (Tiandz Tech- 2. Material and Methods nology, Ltd., Beijing, China) according to the manufacturer’s instruction manual and sequenced by Genscript (Nanjing 2.1. Isolation of Fungal Strains Antagonistic to Different Fungi. Biotech Co., Ltd., China). The ITS rDNA sequences of the Soil samples were collected from Qinling Mountain, Shaanxi strains were compared with available ITS rDNA sequences Province, in China, and air-dried. A one-gram sample was in GenBank databases using the BLAST search facility at added to 9 mL sterile water and cultivated in a shaker at ∘ the National Center for Biotechnology Information (NCBI). 28 C for 1 h. The soil suspension was then serially diluted Sequences retrieved were aligned with the most similar-type with sterile water with 50 𝜇L spread onto Potato Dextrose ∘ strains obtained using CLUSTAL X [13]. Neighbor-joining Agar (PDA) and incubated at 28 Cfor5dayswithpurified (NJ) analysis and editing of the trees were performed using and separated monospore strain. The colonies were streaked Mega 6. onto new PDA plates for purification. The purified colonies were coded and maintained on PDA medium and stored at ∘ 4 C. 2.3. Evaluation of the Growth-Promoting Effect of Strain QLP12 The antagonism assay was conducted against differ- in Greenhouse Conditions ent pathogenic fungi (Botrytis cinerea, Fulvia fulva, Col- letotrichum gloeosporioides, Mucor piriformis, Penicillium 2.3.1. Effects on Eggplant Seed Germination and Bud Growth. expansum, Trichothecium roseum, Rhizoctonia solani, Verti- EggplantcultivarLanzanumber1wasusedingreenhouse cillium dahlia, Fusarium oxysporum f. sp. niveum, and F. and field experiments. After sterilization for 5 minutes in oxysporum f. sp. vasinfectum) by the dual culture technique. ∘ a 1% NaClO solution, eggplant seeds were washed three Ten indicator isolates were grown on PDA plates at 25 Cfor5 times in sterile distilled water (SDW) and air-dried under days. A 5 mm diameter agar disk was placed on the center sterile conditions. The seeds were placed on filter paper in of a PDA plate and an equal amount of biocontrol strains an 18 cm diameter Petri dish which had been soaked for 8 −1 was introduced on both sides of the same plate. All isolates 1 min in the fermentation broth (about 10 cfu⋅mL )and ∘ ∘ were incubated on PDA plates at 25 Cfor3–5days.Then, incubated at 28 Cfor6d.Measurementsofbudlengthsand inhibition zones were measured and inhibition rates were calculations of germination rates followed. The seeds soaked calculated, where IR =(𝐷CK −𝑅𝑇)/𝐷CK, 𝐷CK,and𝑅𝑇 were withliquidsubstratewereusedascontrols(thesamebelow). the size of the inhibited area without and with biocontrol Three Petri dishes were used for each treatment and each strains, respectively. This test was repeated twice and three dish held ten seeds. This experiment was conducted three plates for each treatment were used as replications. The strain times. with the strongest antifungal effect (inhibition zone) was selected for further studies. The strain QLP12 and pathogenic 2.3.2. Colonization in the Rhizosphere and the inside of Egg- fungi growing on PDA or in Potato Dextrose Broth (PDB) at ∘ ∘ plants. Separatedeggplantseedsweresterilizedasdescribed 28 C were stored long term in 20% glycerol at −20 Catthe above (Section 2.3.1) and seedlings at the three true-leaf stage College of Plant Protection, Northwestern A&F University, were transplanted in sterile soil mixed with potting media Shaanxi, China. ∘ (m : m =2:1)andcultivatedinthegreenhouseat25 ± 2 C, 70±5%relativityhumidityand16hlight/8hdark.Root,stem, 2.2. Identification of the Strain QLP12 by Morphology and ITS leaf, and rhizosphere soil (1 g on their own) were sampled, rDNA Gene Sequence Analysis. Preliminary identification of each from 10 plants at 14, 19, 24, and 29 d. The coloniza- the strain QLP12 was based on colony and mycelial morphol- tion study depended on the tolerance of the strain QLP12 ogy, including conidia production and type of conidiophores. to distinctive fungicides. Using the dilution-plate method, BioMed Research International 3

−1 the isolate was isolated from the PDA plate with 100 𝜇g⋅mL true leaf, the plants were transplanted into pots with a 20 : 1 𝜇 ⋅ −1 mixture of soil and fermentation broth of the QLP12 (about 50% carbendazim WP and 50 g mL rifampicin. This pro- 8 −1 6 −1 cedure was followed by calculations of the colony or colonies. 10 cfu⋅mL )dilutedthereafter100times(10 cfu⋅mL )and This experiment was conducted three times. followed by a second treatment with QLP12 fermentation 6 −1 broth (10 cfu⋅mL ) 30 d after seeding transplantation. Mean 2.3.3. Determination of Chlorophyll Content. In greenhouse, plant height, number of leaves, leaf area (calculated by other eggplants were chosen to fulfill this task. An amount squares printed on coordinate paper), plant fresh height, of 0.1 g tested plant material, sampled at 30 d after sowing, root dry weight, and dry weight of leaves and shoot were for example, leaves, was chopped into small pieces in a recorded after 60 d and compared by ANOVA. This treat- mortar and had 0.5 mL acetone, quartz sand, and 10 mL 80% ment that contained 10 pots was replicated 3 times with acetone added and ground into a homogenate. The extract one plant per pot, using treatment with uncolonized sub- was poured into a 25 mL volumetric flask with 80% acetone. strate as controls. Experiments were analyzed independent- 80% acetone served as a blank test. Optical density (OD) ly. was measured spectrophotometrically under 645, 552, 663, and 470 nm. According to the OD data, concentrat