pathogens in the litter. Therefore we cultivated bacteria and fungi from the leaves of the two Prosopis congeners and applied them to two species native to India, Brassica juncea and Chloris dolichostachya. Cultivation of bacterial and fungal communities of Prosopis leaves was achieved by streaking the leaves of each congener (in triplicate) across the surface of several standard microbiological media agar plates. Plates were incubated at 37uC for 1 week in order to observe any potential bacterial or fungal growth. Five different media were used to cultivate microbial growth. The media used to select for bacteria were R2 agar [34], Minimal Agar, and Nutrient Agar [35] treated with 300 mg L21of chloramphenicol to inhibit fungal growth. The media used to select for fungi were Mycobiotic Agar and Potato Dextrose Agar [35]. After the incubation period, individual colonies were selected from the media plates and were used to make separate bacterial and fungal community inocula from each Prosopis congener. Each inoculum (either bacterial or fungal) was prepared by aseptically transferring individual microbial colonies into sterile Eppendorf tubes (2 mL volume) filled with 1.5 mL sterile Milli-Q water, and gently vortexed. Mixing 0.5 mL of the bacterial community inoculum with 0.5 mL of the fungal community inoculum made a third composite microbial inoculum. Individual seedlings of B. juncea and C. dolichostachya were germinated in 200 cm3 rocket pots filled with 20/30 grit silica sand, and after two weeks were inoculated with the bacterial, fungal, or composite microbial inocula, with n = 15 for each species and treatment combination. We measured survival of these seedlings 20 days after inoculation. Chemical Characteristics of Soil Amended with Leaf Leachate of Two Congeners To determine the effect of leaf leachate treatment on soil chemistry, 50g soil in a 9 cm Petri dish was amended with 15 mL distilled water (control) and 1x diluted P. juliflora or P. cineraria leaf leachate (0 and 300 mL P. juliflora or P. cineraria leaf leachate/g soil) as described above. Six replicate Petri dishes were used per treatment. Immediately after treatment, lids of Petri dishes were removed and soil was allowed to air-dry for 48 h. Soil was then analysed for pH, electrical conductivity, organic carbon, PO43–P, and total organic N as described above. We tested the difference among P. juliflora, P. cineraria leaf leachate amended and unamended soil for each soil variable using one-way ANOVA and post-hoc Tukey’s test [30]. Effects of Leaf Leachate and Leaf Litter of Prosopis Congeners on Soil Phenolic Content Soil amended with leaf leachates of P. juliflora or P. cineraria, as described above, was also analyzed for total phenolic concentra- tion in addition to other chemical properties. In a separate experiment, we added 60 mg of air dried leaves of P. juliflora or P. cineraria to 5 g soil and moistened the soil with 2 ml distilled water. Litter-amended soil was incubated at 30–34uC for 0, 1, 2, 3, 4, 6, 8, 10 or 14 d. Each treatment and the control (unamended soil) were replicated four times. Measurement of total phenolics in soils from two experiments was performed as described above. We tested the differences among P. juliflora litter, P. cineraria litter, leaf leachate amendments, and untreated soil for total phenolics using one-way ANOVA and post-hoc Tukey’s test [30]. L-tryptophan Recovery from Leaf Leachate Amended Soil Nakano et al. [27] reported that the allelopathic effects of P. juliflora foliage appeared to be caused by L-tryptophan, thus we suspected it might play a role in the effects we measured for litter and leaf leachates. For this, we quantified L-tryptophan in leaf Invasion of Prosopis juliflora leachates from P. juliflora or P. cineraria and in soil amended with these leachates. Two ml of leaf leachate from P. juliflora or P. cineraria was added to 10 mL methanol, and L-tryptophan was quantified with high-performance liquid chromatography (HPLC). Five g soil was treated with 2 mL leaf leachate from P. juliflora, P. cineraria or with distilled water and then immediately extracted in 10 mL methanol by shaking for 20 min followed by centrifugation at 4500 rpm for 10 min. The supernatant was filtered through 0.2 mm PES syringe filters directly into HPLC vials. L-tryptophan level in methanol extract of each soil and leachate sample was determined by using HPLC (Waters Corp., Milford, U.S.A), employing a Waters Spherisorb 5 mm ODS2 column (4.66250 mm Analytical Column). The mobile phase consisted of 50 mM sodiumphosphate buffer, pH 3.5 containing 20% meth- anol (v/v) and flow rate was 1 mL/min. L-tryptophan was detected at 214 nm using a photodiode array detector. 10–20 mL of sample was injected in the partial loop needle overfill mode, run time for each sample was 20 min, and the retention time of L- tryptophan was 8.17 min with 0.27% RSD. L-tryptophan concentration in leaf leachates from two congeners was compared using independent samples t-test [30]. Results Prosopis juliflora and Species Richness in Native and Non- native Ranges Plant species richness in the understory of P. juliflora in its native range of Venezuela was 48% and 63% higher than in nearby open areas, at Puente Real and Mucumi, respectively (Figure 2, middle panel; Funderstory vs. open = 324.0; df = 1,56; p = 0.035). Plant species richness in the understory of P. pallida in its native range of Peru was 27% and 44% higher than in nearby open areas, at Chulucanas and Catacaos, respectively in 2008 (Figure 2, upper panel; t Chulucanas = 5.13; df = 1,94; p,0.001; t Catacaos = = 2.67; df = 1,92; p = 0.011). However, in 2009 richness was 28% and 25% lower than in the open at those same sites (Figure 2, upper panel; t Chulucanas = 2.70; df = 1,70; p = 0.009; t Catacaos = = 4.678, df = 1, 118, p,0.0001). Analyzed over both years P. pallida had no effect on understory richness in its native range in Peru (Fcanopy = 0.79; df = 1,374; P = 0.778). In the non-native range of India, species richness in P. juliflora invaded areas was 32%, 54% and 78% lower at Delhi, Gujarat, and Rajasthan, respectively, compared to areas without P. juliflora (Figure 2, lower panel: tDelhi = 2.72; df = 1,18, p = 0.014; tGujarat = 3.37; df = 1,12; p = 0.006, and for Rajasthan, Mann- Whitney U test, p = 0.012). The mean effect in India over all three sites was a decrease in species richness of 56%. In habitats invaded by P. juliflora in India native species accounted for 92%, 67% and 100% of the total number of species present below P. juliflora canopies at sites Delhi, Rajasthan and Gujarat, respectively. In Honolulu, Hawaii species richness in P. juliflora invaded areas was 70% and 65.3% lower at Sand Island and Iroquis point, respectively (Figure 2, lower panel: tSand Island = 5.93, df = 1,18, p,0.001; tIroquis point = 7.49; df = 1,18, p,0.001). In Hawaii, however, more than 90% of understory species were non-native. Effects of Prosopis Congeners on Species Richness At a fourth site in India, species richness did not differ beneath canopies of P. cineraria relative to open areas, but species richness was 63% lower under P. juliflora canopies than in the open and 50% lower than under P. cineraria canopies (Figure 3; F = 10.018; df = 2,27; P,0.001). PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44966