out HWT served as control. For the

out HWT served as control. For the enzyme assay, fruit samples
were obtained from 10 fruits containing the pericarp and flesh at
0, 1, 2, 3, 4 and 5 days after treatment (DAT). Each treatment contained
three replicates and the experiment was repeated twice.
PAL was extracted by the method of Zhao et al. (2008), with some
modification. Tissue sample (10 g) was mixed with 4 mL of ice-cold
sodium borate buffer (100 mM, pH 8.7) and ground thoroughly at
4 C. The homogenate was centrifuged at 17,000g for 30 min at
4 C, and the resulting supernatant was collected for the enzyme assay.
PAL activity was analyzed using the method of Assis et al.
(2001), with some modification. One milliliter of enzyme extract
was incubated with 2 mL of borate buffer and 0.5 mL of L-phenylalanine
(20 mM) for 60 min at 37 C. The reaction was stopped with
0.1 mL 6 mol L1 HCl. PAL activity was determined by the production
of cinnamate, measured by the absorbance change at 290 nm.
The blank was the crude enzyme preparation mixed with L-phenylalanine
with zero time incubation. The specific enzyme activity was
defined as nmol cinnamic acid h1 mg1 of protein.
For chitinase (CHI) and b-1,3-glucanase, enzymes were extracted
according to Yao and Tian (2005). Tissue samples (10 g)
with 0.3 g polyvinyl polypyrrolidone (PVPP) were ground with
30 mL sodium acetate buffer (50 mmol l1
, pH 5.0) at 4 C. The
homogenate was centrifuged at 17,000g for 30 min at 4 C, and
the resulting supernatant was collected for the enzyme assay.
CHI activity was determined using the method of Wirth and
Wolf (1990), with slight modification. CHI activity was measured
by mixing 1 mL of crude enzyme solution with 2 mL of 2% dye-labeled
carboxymethyl chitin in 50 mmol l1 sodium acetate buffer
(pH 5.0). After 1 h of incubation at 37 C, the reaction was stopped
by adding 1 mL of 1 mol L1 HCl, the reaction mixture was cooled
and centrifuged. The absorbance of the supernatant was measured
at 550 nm. The specific enzyme activity was expressed as lmol
product h1 mg1 protein.
b-1,3-Glucanase activity was assayed by measuring the amount
of reducing sugar released from the substrate by the dinitrosalicylate
method (Ippolito et al., 2000), with some modification. A total
volume of 250 lL of enzyme preparation was incubated with
250 lL of 0.5% laminarin (w/v) for 1 h at 37 C. Two hundred microliters
of sterile distilled water was added to 50 lL of the reaction
mixture. The blank was the crude enzyme preparation mixed with
laminarin with zero time incubation. The reaction was stopped by
adding 250 lL of 3,5-dinitrosalicylate and boiling for 5 min in a
water bath. The solution was diluted with 4 mL of distilled water
and the amount of reducing sugars was measured at 500 nm. The
specific activity of b-1,3-glucanase was expressed as the formation
of 1 lmol glucose equivalents h1 mg1 protein.
Protein content was determined according to Bradford (1976)
with bovine serum albumin (Sigma Chemicals Co., St. Louis, USA)
as standard.
2.9. Statistical analysis
The results from three independent experiments were accordant,
and data from one representative experiment are presented
in this paper. All statistical analyses were performed with SPSS version
13.0 (SPSS, Inc., Chicago, IL, USA). Data from assays of population
dynamics and enzyme activities were compared in a Student’s
t-test. Others were analyzed by one-way ANOVA. Mean separations
were performed by Duncan’s multiple range tests. Differences
at P < 0.05 were considered significant.
3. Results
3.1. Efficacy of hot water treatment on control of gray mold in tomato
fruit
Disease incidence of gray mold in hot water treated fruit was
significantly lower than that of the control (P < 0.05), except for
the fruit treated for the longest time (60 min) (Fig. 1A). Moreover,
HWT at all treatment time significantly reduced lesion diameter
(P < 0.05) (Fig. 1B). The best inhibition of this disease was achieved
when the fruit were treated with hot water at 42 C for 40 min.
Therefore, HWT for 40 min was chosen for further study to evaluate
the effect on disease control of the combination with antagonistic
yeasts, the effect on population dynamics of the yeasts and
on induction of defense-related enzyme activities of fruit.
3.2. Effects of yeasts in combination with hot water treatment on
control of gray mold in tomato fruit
Disease incidence and lesion diameter in all treated fruit were
significantly lower than those of the control fruit (P < 0.05)
(Fig. 2). HWT (40 min), C. guilliermondii and P. membranaefaciens,
as stand-alone treatments, reduced the disease incidence from
81.7% (control) to 61.7%, 40.0% and 45.0%, respectively. The combination
of the two yeasts and HWT exhibited a synergistic effect,
which decreased the disease incidence to a lower level, 21.7% for
C. guilliermondii and 26.7% for P. membranaefaciens, respectively.

Kết quả (Việt) 1:

ra HWT phục vụ như là kiểm soát. Cho khảo nghiệm enzym, trái cây mẫuđã thu được từ 10 loại trái cây có chứa các pericarp và thịt lúc0, 1, 2, 3, 4 và 5 ngày sau khi điều trị (DAT). Mỗi lần điều trị bao gồmba sao chép và thử nghiệm được lặp đi lặp lại hai lần.PAL đã chiết xuất theo phương pháp của Triệu Tử Dương et al. (2008), với một sốSửa đổi. Mô mẫu (10 g) được trộn lẫn với 4 mL lạnhbộ đệm borat natri (100 mM, độ pH 8.7) và đất triệt để tại4 C. Homogenate ly 17.000 g trong 30 phút tại4 C, và supernatant quả được thu thập cho khảo nghiệm enzym.PAL hoạt động đã được phân tích bằng cách sử dụng các phương pháp của Assis et al.(2001), với một số sửa đổi. Một trong những milliliter chiết xuất men tiêu hóađược ủ với 2 mL borat đệm và cách 0.5 mL L-nmol(20 mM) trong 60 phút ở 37 C. Các phản ứng đã được dừng lại với0.1 mL 6 mol L1 HCl. PAL hoạt động đã được xác định bởi việc sản xuấtcủa cinnamate, được đo bằng cách thay đổi hấp thu tại 290 nm.Chỗ trống là chuẩn bị thô enzyme hỗn hợp với L-nmolvới số thời gian ấp trứng. Hoạt động của enzyme cụ thể làđịnh nghĩa là phenylalanine cinnamic acid h1 mg1 của protein.Chitinase (CHI) và b-1,3-glucanase, enzyme đã chiết xuấttheo Yao và Tian (2005). Mẫu mô (10 g)với cách 0.3 g polyvinyl polypyrrolidone (PVPP) đã được nghiền với30 mL natri axetat đệm (50 mmol l1pH 5,0) tại 4 C. cáchomogenate ly 17.000 g trong 30 phút tại 4 C, vàsupernatant kết quả đã được thu thập cho khảo nghiệm enzym.Hoạt động CHÍ đã được xác định bằng cách sử dụng phương pháp Wirth vàSói (1990), với sửa đổi chút ít. CHI hoạt động được đobằng cách trộn 1 mL dầu thô enzyme giải pháp với 2 mL 2% có nhãn thuốc nhuộmCarboxymethyl chitin trong 50 mmol l1 natri axetat đệm(pH 5,0). Sau 1 h ấp ở 37 C, các phản ứng đã được ngừng lạibằng cách thêm 1 mL 1 mol L1 HCl, phản ứng hỗn hợp được làm mát bằng nướcvà ly. Hấp thu supernatant đã được đotại 550 nm. Hoạt động của enzyme cụ thể được biểu thị dưới dạng lmolsản phẩm h1 mg1 protein.b-1,3-Glucanase hoạt động được assayed bằng cách đo lượnggiảm đường phát hành từ bề mặt của dinitrosalicylatephương pháp (Ippolito et al., 2000), với một số sửa đổi. Tổng cộngkhối lượng 250 lL enzyme chuẩn bị được ủ với250 lL laminarin 0.5% (w/v) cho 1 h 37 c. Hai trăm microlitersnước cất vô trùng đã được thêm vào 50 lL của phản ứnghỗn hợp. Chỗ trống là enzym thô chuẩn bị pha trộn vớilaminarin với số không thời gian ấp trứng. Các phản ứng đã dừng lạiThêm 250 lL 3,5-dinitrosalicylate và đun sôi trong 5 phút trong mộtnước tắm. Các giải pháp đã được pha loãng với 4 mL nước cấtvà số tiền giảm đường được đo ở 500 nm. CácCác hoạt động cụ thể của b-1,3-glucanase đã được thể hiện như là sự hình thành1 lmol glucose tương đương h1 mg1 protein.Protein nội dung đã được xác định theo Bradford (1976)with bovine serum albumin (Sigma Chemicals Co., St. Louis, USA)as standard.2.9. Statistical analysisThe results from three independent experiments were accordant,and data from one representative experiment are presentedin this paper. All statistical analyses were performed with SPSS version13.0 (SPSS, Inc., Chicago, IL, USA). Data from assays of populationdynamics and enzyme activities were compared in a Student’st-test. Others were analyzed by one-way ANOVA. Mean separationswere performed by Duncan’s multiple range tests. Differencesat P < 0.05 were considered significant.3. Results3.1. Efficacy of hot water treatment on control of gray mold in tomatofruitDisease incidence of gray mold in hot water treated fruit wassignificantly lower than that of the control (P < 0.05), except forthe fruit treated for the longest time (60 min) (Fig. 1A). Moreover,HWT at all treatment time significantly reduced lesion diameter(P < 0.05) (Fig. 1B). The best inhibition of this disease was achievedwhen the fruit were treated with hot water at 42 C for 40 min.Therefore, HWT for 40 min was chosen for further study to evaluatethe effect on disease control of the combination with antagonisticyeasts, the effect on population dynamics of the yeasts andon induction of defense-related enzyme activities of fruit.3.2. Effects of yeasts in combination with hot water treatment oncontrol of gray mold in tomato fruitDisease incidence and lesion diameter in all treated fruit weresignificantly lower than those of the control fruit (P < 0.05)(Fig. 2). HWT (40 min), C. guilliermondii and P. membranaefaciens,as stand-alone treatments, reduced the disease incidence from81.7% (control) to 61.7%, 40.0% and 45.0%, respectively. The combinationof the two yeasts and HWT exhibited a synergistic effect,which decreased the disease incidence to a lower level, 21.7% forC. guilliermondii and 26.7% for P. membranaefaciens, respectively.