Pol. J. Environ. Stud. Vol. 24, No. 6 (2015), 2515-2524 DOI: 10.15244/pjoes/59985 Original Research Effect of Scots Pine Sawdust Amendment on Abundance and Diversity of Culturable Fungi in Soil Monika Małecka1, Hanna Kwaśna2* 1Forest Research Institute, Department of Forest Protection, Sękocin Stary, Braci Leśnej 3, 05-090 Raszyn, Poland 2Department of Forest Pathology, Poznań University of Life Sciences, Wojska Polskiego 71c, 60-625 Poznan, Poland Received: August 10, 2015 Accepted: September 30, 2015 Abstract Scots pine and birch were planted in soils left fallow for 3, 6, or 15 years after arable cropping. We inves- tigated the effects of Scots pine sawdust amendment, applied a year before planting, on the abundance and diversity of culturable soil fungi 4, 14, and 16 years after treatment. The treatment was intended to increase populations of fungi antagonistic to Heterobasidion and soil suppressiveness to tree root pathogens. Effects of treatment on fungal abundance were inconsistent; general or local, seasonal or continuous decreases or increas- es, often significant, were observed. There were, however, significant and continuous increases in frequency of antagonistic Clonostachys + Trichoderma and the mycorrhizal fungus Oidiodendron in treated soils compared with the control in all three fallow areas. Local and seasonal decreases in frequency of Penicillium + Talaromyces, Pseudogymnoascus, and entomopathogenic and nematophagous species were observed in treat- ed soils. Abundance of fungi was moderately and negatively correlated with soil pH (R2 = -0.61, P<0.0001). Abundance of Clonostachys + Trichoderma was moderately and positively correlated with mean annual tem- perature and positively correlated with total annual rainfall. Fresh sawdust, even applied undecomposed and without added mineral N (to aid microbial decomposition and plant growth), may be beneficial in sandy soil. Keywords: Heterobasidion, Oidiodendron, organic matter, soil fungi, Trichoderma Introduction Heterobasidion species are considered to be the most economically important conifer forest pathogens in the In 2002 the National Program for the Augmentation of northern hemisphere [1, 2]. Scots pine becomes seriously Forest Cover in Poland was revised. The Program antici- damaged by Heterobasidion annosum (Fr.) Bref. [3]. pates afforestation of 1.5 million ha of marginal land The fungus spreads through aerial basidiospores to stump (including post-agricultural and fallow land) and an surfaces and wounds, and by mycelia via root contacts from increase in forest cover to 30% by 2020 and 33% by 2050. tree to tree. It causes root and butt rot, growth loss, and mor- Considering soil and weather conditions, this marginal land tality of trees. In favourable conditions seedlings can is being planted with Scots pine (Pinus sylvestris L.), which become infected very soon after planting. In Europe and the is the dominant forest tree species in Poland (71%). USA Heterobasidion is responsible for the loss of $1.5-2 billion annually. *e-mail: [email protected] 2516 Małecka M., Kwaśna H. Damage is generally greater in the first or second gen- left under fallow for 15 years was sandy clay loam (sand erations of trees grown on post-agricultural or fallow land, 77%, clay 13%, silt 10%). The soils were analysed chemi- which has the least organic matter; a deficiency in nitrogen, cally at the Forest Research Institute in Sękocin Stary. KCl potassium, phosphorus, and magnesium; higher pH; was analysed with a potentiometer according to norm PN- increased soil compaction below the plough level; and poor ISO 103390:1997. Organic carbon was analysed according capillarity for water [4]. The shortage of lignified tissues to norm PN-ISO 10694, total nitrogen according to norm and a specific carbon and energy source for microbiota cre- PN-13878, extractable phosphorus with the Egner-Riehm ates an unfavourable microbiological habitat and causes method, and extractable potassium, magnesium, calcium, deficiency of microorganisms that are potentially competi- and sodium according to procedure PB-05 ed. 2. The soil tive and antagonistic toward H. annosum and helpful in for- pH in H2O was 4.4-7.0, organic carbon was 0.87-2.39%, est site-forming processes. and total nitrogen content was 0.059-0.1% (Table 1). The aim of our study was to evaluate the abundance, Mean seasonal and annual temperatures in Głęboki Bród in diversity, and temporal and spatial distribution of culturable 1999, 2009, and 2011 were higher than in 1995 and higher fungi in soils left fallow for different lengths of time, then (by 4-26%) than the average (Table 2). Total seasonal and amended with fresh Scots pine sawdust. The hypothesis annual rainfall was similar to the average. was that the organic matter amendment would change the physical and chemical properties of the soil, which would Isolation and Identification of Fungi in turn initiate and stimulate microbiological shifts that would increase the population of fungal antagonists of The classical soil-dilution method was applied for qual- Heterobasidion and suppressiveness of soil to root itative and quantitative analyses of fungal communities in pathogens. Studies made 4, 14, and 16 years after treatment 1995 (when nucleic acid sequencing was not easily acces- were intended to show the temporal dynamics and diversi- sible), and 4, 14, and 16 years later (for direct comparison ty of microorganisms. with results from 1995). The affiliation, activity, and micro- This study is part of a larger project on development and biological interactions of many culturable soil fungi are promotion of non-chemical methods of pest management already known and can be used for evaluation of the possi- as part of an integrated pest management strategy. ble microbiological status of soil (i.e., comparing soil qual- ities and properties). A subsample (1 g) of soil from each treatment was dilut- Material and Methods ed in 149 g of sterile quartz sand in a flask, in a safety cab- inet. Three replicates (= three flasks) of soil-sand mixture Site Description, Treatments, and Sampling were prepared for each treatment and each control. A sub- sample (0.26 mg) of the soil-sand mixture was put into a The experiment was established in three areas (each Petri dish and covered with liquid (50ºC) Johnson-Martin’s 300 m2, 1 km apart), that had been left fallow for 3, 6, and agar (JMA). Thirty replicates were made from the three 15 years in Głęboki Bród Forest District in northeast Poland flasks for each treatment and each control. All plates were (geographic coordinates 53.983333, 23.3). In October 1995, incubated for 20 days at 25ºC. All colonies on each plate four treatment plots and four control plots (each 25 m2) were were examined macro- and microscopically and distin- designated at the four corners of each area. The first soil guished on the basis of color, growth rate, hyphal charac- samples were collected in October 1995; six soil cores teristics, and sporulation. Colonies of each species were (400 g each) were taken from each corner of each plot. counted and representatives of fungi were identified by The upper and lower 5 cm of each core were discarded and morphotyping on potato dextrose agar (PDA) and synthet- the central 10 cm retained, bulked, and mixed by rotating ic nutrient agar (SNA). Aspergillus and Penicillium species for 24 h. The soil was afterward stored at 4ºC for 24 hours. were identified on Czapek yeast autolysate agar (CYA) and Two days after sampling the soil was subjected to chemical 2% malt extract agar (MEA). Identification was made using and mycological analyses. One day after sampling, a 5-cm- mycological keys [5-8]. thick layer (9 kg·m-2) of fresh Scots pine sawdust was Abundance of fungi was defined as the number of colony spread onto the treatment plots. All treatment plots and non- forming units (cfu) in a sample. Frequency of an individual treated control plots were next ploughed to a depth of species or group of species was defined as proportion (%) of 35 cm. In spring 1996 the soil was again ploughed and har- isolates in the total number of isolates. Diversity was defined rowed. Scots pine and birch seedlings were then planted in as the number of species in a sample. A species, or group of all plots (one plant per 1 m2). (The sawdust was fresh and related species of fungi, was considered as: non-composted and came from tree cutting that took place i) eudominant, with frequency >10% in 1995.) ii) dominant, with frequency 5-10% Further soil samples were collected 4, 14, and 16 years iii) subdominant, with frequency 2-5% later (in October 1999, 2009, and 2011) and subjected to the iv) recedent, with frequency 1-2% same chemical and mycological analyses that were carried v) subrecedent, with frequency < 1% (in at least one treat- out by the same employees as in 1995. ment). The soils left under fallow for 3 and 6 years were Fungi in the main dominance class were often grouped defined as sand (sand 89-91%, clay 4-7%, silt 4%). The soil on the basis of their morphology and taxonomy. Effect ofScotsPineSawdust... 2517 Table 1. Chemical properties of soils left fallow for different durations before (1995) and after (1999, 2009, 2011) sawdust amendment and tree planting. 3 years fallow 6 years fallow 15 years fallow Soil characteristics 1999 2009 2011 1999 2009 2011 2009 2011 1995 1995 1995 S C S C S C S C S C S C S C S C pH in KCl 3.8 5.0 4.9 4.85* 4.92* 5.23 4.33 6.0 4.3 4.2 4.89* 4.15* 4.3 4.0 4.1 4.39* 4.18* 4.1 4.1 F=16.56 F= 8.03 P=0.8968 P=0.0969 P=0.0066